Method of using adeno-associated virus with variant capsid

ABSTRACT

Provided herein are variant adeno-associated virus (AAV) capsid proteins having one or more modifications in amino acid sequence relative to a parental AAV capsid protein, which, when present in an AAV virion, confer increased infectivity of one or more types of retinal cells as compared to the infectivity of the retinal cells by an AAV virion comprising the unmodified parental AAV capsid protein. Also provided are recombinant AAV virions and pharmaceutical compositions thereof comprising a variant AAV capsid protein as described herein, methods of making these rAAV capsid proteins and virions, and methods for using these rAAV capsid proteins and virions in research and in clinical practice, for example in, e.g., the delivery of nucleic acid sequences to one or more cells of the retina for the treatment of retinal disorders and diseases.

This application is a divisional of U.S. patent application Ser. No.16/300,446 filed Nov. 8, 2018 which pursuant to 35 U.S.C. § 119(e),claims priority to the filing date of U.S. Provisional PatentApplication Ser. No. 62/336,441 filed May 13, 2016; U.S. ProvisionalPatent Application Ser. No. 62/378,106 filed Aug. 22, 2016; U.S.Provisional Patent Application Ser. No. 62/384,590 filed Sep. 7, 2016;U.S. Provisional Patent Application Ser. No. 62/454,612 filed Feb. 3,2017; the full disclosures of which are herein incorporated byreference.

INCORPORATION BY REFERENCE OF A SEQUENCE LISTING PROVIDED AS A TEXT FILE

A Sequence Listing is provided herewith as a text file, “4D_ST25.txt.”created on May 12, 2017 and having a size of 198 KB. The contents of thetext file are incorporated by reference herein in their entirety.

FIELD OF THE INVENTION

The invention disclosed herein relates generally to the field ofadeno-associated virus (AAV) virions comprising variant capsid proteinsand the generation of such variant capsids using directed evolutiontechniques.

BACKGROUND OF THE DISCLOSURE

Inherited retinal diseases encompass a large group of heterogenousgenetic diseases that affect approximately 1 in 3000 people (greaterthan 2 million people worldwide) and are a major source of severe visionloss or blindness. Complex, multifactoral retinal diseases such as wetage related macular degeneration (wAMD) and diabetic retinopathy (DR)impact even more individuals, with 1.7 million Americans currentlyliving with severe central vision loss associated with wAMD and almostone-third of adults over age 40 years with diabetes being visuallyimpaired. These diseases are typically associated with the dysfunctionor death of one or more types of cell of the retina, in some instancesdue to the absence of expression or function of a key protein, e.g.RPE65 in LCA2, in other instances due to gene mutations that createtoxic gene products, e.g dominant mutations that affect rhodopsinprotein folding, or in yet other instances due to changes in retinalphysiology induced by the ectopic expression of a protein, e.g. VEGF inwAMD.

One approach to addressing this great unmedical need is gene-basedadeno-associated virus (AAV)-mediated therapy, in which a recombinantadeno associated virus (rAAV) is used to deliver a gene to one or moretypes of cells in the retina, for example to replace a missing gene, tocorrect a dominant defective gene, or to provide a template forcontinuous protein therapy. While AAV-based clinical gene therapy hasbeen increasingly successful, it is still fraught with shortcomings withregard to viral vector properties, including, for example, targeting thedesired cells of the retina with high efficiency. For example, multiplehomologous primate AAV serotypes and numerous nonhuman primate serotypeshave been identified and characterized, with AAV2 being the bestcharacterized among the AAV serotypes and the first to be adapted as agene delivery vehicle in the eye. However, these AAVs (including AAV2)have not been reported to be effective at transducing the deeper celltypes of the retina when delivered via intravitreal administration.Accordingly, there is a need in the art for new AAV variants withsuperior transduction capabilities that will provide for more effectivegene-based delivery to the cells of the retina for the treatment ofocular disease. There is a need in the art for such AAV variants whichexhibit an enhanced retinal transduction profile—in some instancesbroadly, in other instances preferentially to certain retinal celltypes—as compared to wild-type AAVs and AAV variants as known in theart.

Naturally occurring AAV is a single stranded DNA virus that containsthree open reading frames, rep, cap, and aap. The first gene, rep,encodes four proteins necessary for genome replication (Rep78, Rep68,Rep52, and Rep40), the second, cap, expresses three structural proteins(VP1-3) that assemble to form the viral capsid, and the third expressesthe assembly activating protein (AAP) that is essential for capsidassembly. AAV is dependent upon the presence of a helper virus, such asan adenovirus or herpesvirus, for active replication. In the absence ofa helper virus, AAV establishes a latent state in which its genome ismaintained episomally or integrated into the host chromosome in theAAVS1 locus.

In vitro and in vivo-directed evolution techniques may be used to selectfor AAV variants that offer an improvement over current AAV-based genedelivery vectors. Such directed evolution techniques are known in theart and described, e.g., in PCT publication WO 2014/194132 and Kotterman& Schaffer (Nature Review Genetics, AOP, published online 20 May 2014;doi: 10.1038/nrg3742), both of which are incorporated herein in theirentirety by reference. Directed evolution is a capsid engineeringapproach that emulates natural evolution through iterative rounds ofgenetic diversification and selection processes, thereby enabling theaccumulation of beneficial mutations that progressively improve thefunction of a biomolecule such as an AAV-based virion. In this approach,wild-type AAV cap genes are diversified to create large geneticlibraries that are packaged to generate libraries of viral particles,and selective pressure is applied to isolate unique variants withsuperior phenotypes that can overcome gene delivery barriers.

AAV variants have been disclosed in, for example, in U.S. Pat. Nos.9,193,956; 9,186,419; 8,632,764; 8,663,624; 8,927,514; 8,628,966;8,263,396; 8,734,809; 8,889,641; 8,632,764; 8,691,948; 8,299,295;8,802,440; 8,445,267; 8,906,307; 8,574,583; 8,067,015; 7,588,772;7,867,484; 8,163,543; 8,283,151; 8,999,678; 7,892,809; 7,906,111;7,259,151; 7,629,322; 7,220,577; 8,802,080; 7,198,951; 8,318,480;8,962,332; 7,790,449; 7,282,199; 8,906,675; 8,524,446; 7,712,893;6,491,907; 8,637,255; 7,186,522; 7,105,345; 6,759,237; 6,984,517;6,962,815; 7,749,492; 7,259,151; and 6,156,303; United StatesPublication Numbers 2013/0295614; 2015/0065562; 2014/0364338;2013/0323226; 2014/0359799; 2013/0059732; 2014/0037585; 2014/0056854;2013/0296409; 2014/0335054 2013/0195801; 2012/0070899; 2011/0275529;2011/0171262; 2009/0215879; 2010/0297177; 2010/0203083; 2009/0317417;2009/0202490; 2012/0220492; 2006/0292117; and 2004/0002159; EuropeanPublication Numbers 2692731 A1; 2383346 B1; 2359865 B1; 2359866 B1;2359867 B1; and 2357010 B1; 1791858 B1; 1668143 B1; 1660678 B1; 1664314B1; 1496944 B1; 1456383 B1; 2341068 B1; 2338900 B1; 1456419 B1; 1310571B1; 1456383 B1; 1633772 B1; and 1135468 B1; and International (PCT)Publication Numbers WO 2014/124282; WO 2013/170078; WO 2014/160092; WO2014/103957; WO 2014/052789; WO 2013/174760; WO 2013/123503; WO2011/038187; and WO 2008/124015; WO 2003/054197; however, none of thesereferences disclose the embodiments and/or features and/or compositionof matter structures of the AAV variants disclosed herein.

All documents and references cited herein and in the referenced patentdocuments, are hereby incorporated herein by reference.

SUMMARY OF THE INVENTION

Provided herein are variant adeno-associated virus (AAV) capsid proteinshaving one or more modifications in amino acid sequence relative to aparental AAV capsid protein, which, when present in an AAV virion,confer increased infectivity of one or more types of retinal cells ascompared to the infectivity of the retinal cells by an AAV virioncomprising an unmodified parental AAV capsid protein. Also provided arerecombinant AAV virions and pharmaceutical compositions thereofcomprising a variant AAV capsid protein as described herein, methods ofmaking variant rAAV capsid proteins and virions, and methods for usingthese rAAV capsid proteins and virions in research and in clinicalpractice, for example in the delivery of nucleic acid sequences to oneor more cells of the retina for the treatment of retinal disorders anddiseases.

In some aspects of the disclosure, variant adeno-associated virus (AAV)capsid proteins are provided, these variant AAV capsid proteins havingone or more modifications in amino acid sequence relative to a parentalAAV capsid, which, when present in an AAV virion, confer increasedinfectivity of one or more types of retinal cells (e.g. a photoreceptorcell (e.g. rods; cones), a retinal ganglion cell (RGC), a glial cell(e.g. a Müller glial cell, a microglial cell), a bipolar cell, anamacrine cell, a horizontal cell, and/or a retinal pigmented epithelium(RPE) cell) as compared to the infectivity of the retinal cells by anAAV virion comprising a parental AAV capsid protein that does notcomprise the amino acid sequence modification.

In some aspects of the disclosure, recombinant AAV (rAAV) virions areprovided, these rAAV virions comprising a variant capsid protein asdescribed herein, wherein the rAAV virions exhibit increased infectivityof one or more types of retinal cells (e.g. a photoreceptor cell (e.g.rods; cones), a retinal ganglion cell (RGC), a glial cell (e.g. a Müllerglial cell, a microglial cell), a bipolar cell, an amacrine cell, ahorizontal cell, and/or a retinal pigmented epithelium (RPE) cell)relative to the infectivity of the retinal cell by an AAV virioncomprising a corresponding unmodified parental AAV capsid protein. Insome embodiments, the rAAV virion exhibits increased infectivity of allretinal cells relative to the AAV virion comprising the parental AAVcapsid protein. In other embodiments, the rAAV virion exhibits increasedinfectivity of certain cell types of the retina but not others relativeof the AAV virion comprising the parental AAV capsid protein. Putanother way, the rAAV virion exhibits increased infectivity that ispreferential for certain cell types of the retina but not others, e.g.the rAAV demonstrates a preferentially increased infectivity of one ormore cell types selected from photoreceptor cells, retinal ganglioncells, glial cells, bipolar cells, amacrine cells horizontal cell,and/or retinal pigmented epithelium (RPE) cell, but does not demonstrateincreased infectivity of all cell types.

In some embodiments, the rAAV virion comprises a heterologous nucleicacid. In some such embodiments, the heterologous nucleic acid encodes anRNA that encodes a polypeptide. In other such embodiments, theheterologous nucleic acid sequence encodes an RNA that does not encode apolypeptide, e.g. the heterologous nucleic acid sequence an RNAinterference agent, a guide RNA for a nuclease, etc.

Also provided herein are pharmaceutical compositions comprising thesubject infectious rAAV virions and a pharmaceutically acceptablecarrier.

Also provided is the use of an rAAV virion comprising a variant capsidprotein as herein described in a method of delivering a heterologousnucleic acid to a target cell (such as a retinal cell) by contacting thetarget cell with the rAAV virion. In some embodiments, the target cellis in vivo, such as in the eye of an individual in need of treatment foran ocular disease. In other embodiments, the target cell is in vitro.

Also provided are methods of treating an ocular disease by administeringto a subject in need of such treatment an effective amount of rAAVvirions comprising a variant capsid protein as herein described or apharmaceutical composition comprising an effective amount of the rAAVvirions.

Also provided is an isolated nucleic acid comprising a sequence encodinga variant AAV capsid protein as described herein and a host cellcomprising the isolated nucleic acid. In yet other embodiments, theisolated nucleic acid and/or isolated host cell comprises the rAAV.

In some aspects, the variant AAV capsid protein comprises an insertionof from about 5 amino acids to about 20 amino acids (a “heterologouspeptide”, or “peptide insertion”) in the GH-loop of the capsid protein,relative to a corresponding parental AAV capsid protein, wherein thevariant capsid protein, when present in an AAV virion, confers increasedinfectivity of a retinal cell compared to the infectivity of a retinalcell by an AAV virion comprising the corresponding parental AAV capsidprotein. In some embodiments, the peptide comprises the sequenceselected from the group consisting of QADTTKN (SEQ ID NO:13), ISDQTKH(SEQ ID NO:14), ASDSTKA (SEQ ID NO:15), NQDYTKT (SEQ ID NO:16), HDITKNI(SEQ ID NO:17), HPDTTKN (SEQ ID NO:18), HQDTTKN (SEQ ID NO:19), NKTTNKD(SEQ ID NO:20), ISNENEH (SEQ ID NO:21), QANANEN (SEQ ID NO:22), GKSKVID(SEQ ID NO:23), TNRTSPD (SEQ ID NO:24), PNSTHGS (SEQ ID NO:25), KDRAPST(SEQ ID NO:26), LAQADTTKNA (SEQ ID NO:27), LAISDQTKHA (SEQ ID NO:28),LGISDQTKHA (SEQ ID NO:29), LAASDSTKAA (SEQ ID NO:30), LANQDYTKTA (SEQ IDNO:31), LAHDITKNIA (SEQ ID NO:32), LAHPDTTKNA (SEQ ID NO:33), LAHQDTTKNA(SEQ ID NO:34), LANKTTNKDA (SEQ ID NO:35), LPISNENEHA (SEQ ID NO:36),LPQANANENA (SEQ ID NO:37), LAGKSKVIDA (SEQ ID NO:38), LATNRTSPDA (SEQ IDNO:39), LAPNSTHGSA (SEQ ID NO:40) and LAKDRAPSTA (SEQ ID NO:41). In someembodiments, the peptide consists essentially of the sequence selectedfrom the group consisting of QADTTKN (SEQ ID NO:13), ISDQTKH (SEQ IDNO:14), ASDSTKA (SEQ ID NO:15), NQDYTKT (SEQ ID NO:16), HDITKNI (SEQ IDNO:17), HPDTTKN (SEQ ID NO:18), HQDTTKN (SEQ ID NO:19), NKTTNKD (SEQ IDNO:20), ISNENEH (SEQ ID NO:21), QANANEN (SEQ ID NO:22), GKSKVID (SEQ IDNO:23), TNRTSPD (SEQ ID NO:24), PNSTHGS (SEQ ID NO:25), KDRAPST (SEQ IDNO:26), LAQADTTKNA (SEQ ID NO:27), LAISDQTKHA (SEQ ID NO:28), LGISDQTKHA(SEQ ID NO:29), LAASDSTKAA (SEQ ID NO:30), LANQDYTKTA (SEQ ID NO:31),LAHDITKNIA (SEQ ID NO:32), LAHPDTTKNA (SEQ ID NO:33), LAHQDTTKNA (SEQ IDNO:34), LANKTTNKDA (SEQ ID NO:35), LPISNENEHA (SEQ ID NO:36), LPQANANENA(SEQ ID NO:37), LAGKSKVIDA (SEQ ID NO:38), LATNRTSPDA (SEQ ID NO:39),LAPNSTHGSA (SEQ ID NO:40) and LAKDRAPSTA (SEQ ID NO:41). In someaspects, the variant AAV capsid protein comprises one or more amino acidsubstitutions relative to a corresponding parental AAV capsid protein,wherein the variant capsid protein, when present in an AAV virion,confers increased infectivity of a retinal cell compared to theinfectivity of a retinal cell by an AAV virion comprising thecorresponding parental AAV capsid protein.

In related aspects, the variant AAV capsid protein comprises a peptideinsertion and one or more amino acid substitutions relative to acorresponding parental AAV capsid protein, wherein the variant capsidprotein, when present in an AAV virion, confers increased infectivity ofa retinal cell compared to the infectivity of a retinal cell by an AAVvirion comprising the corresponding parental AAV capsid protein.

Also disclosed herein is a variant AAV capsid protein comprising theheterologous peptide LAISDQTKHA (SEQ ID NO:28) and a P34A substitutionrelative to AAV2.

Also disclosed herein is a variant AAV capsid protein comprising theheterologous peptide LAISDQTKHA (SEQ ID NO:28) and amino acidsubstitutions N312K, N449D, N551S, I698V, and L735Q relative to AAV2.

Also disclosed herein are methods for manufacture and/or delivery of anrAAV comprising a variant AAV capsid as disclosed herein. In addition,provided herein are kits comprising an rAAV comprising a variant AAVcapsid as disclosed herein and for use in methods described herein.

In other embodiments, the AAV virion comprising the variant capsidprotein in the preceding paragraphs may incorporate any of the precedingor subsequently disclosed embodiments. Indeed, it is appreciated thatcertain features of the invention, which are, for clarity, described inthe context of separate embodiments, may also be provided in combinationin a single embodiment. Conversely, various features of the invention,which are, for brevity, described in the context of a single embodiment,may also be provided separately or in any suitable sub-combination. Allcombinations of the embodiments pertaining to the invention arespecifically embraced by the invention and am disclosed herein just asif each and every combination was individually and explicitly disclosed.In addition, all sub-combinations of the various embodiments andelements thereof are also specifically embraced by the invention and aredisclosed herein just as if each and every such sub-combination wasindividually and explicitly disclosed herein.

The Summary of the Invention is not intended to define the claims nor isit intended to limit the scope of the invention in any manner.

Other features and advantages of the invention disclosed herein will beapparent from the following Figures, Detailed Description, and theClaims.

BRIEF DESCRIPTION OF THE FIGURES

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. The patent orapplication file contains at least one drawing executed in color. Copiesof this patent or patent application publication with color drawing(s)will be provided by the Office upon request and payment of the necessaryfee. It is emphasized that, according to common practice, the variousfeatures of the drawings are not to-scale. On the contrary, thedimensions of the various features are arbitrarily expanded or reducedfor clarity. Included in the drawings are the following figures.

FIG. 1 depicts embodiments of a directed evolution methodology. Step (a)depicts the generation of a viral capsid library comprising combinationsof DNA mutation techniques and cap genes. Step (b) depicts the packagingof the viruses such that each viral particle is composed of a mutantcapsid surrounding the cap gene encoding that capsid and purified. Thecapsid library is then placed under selective pressure in vitro or invivo. In this aspect of the directed evolution technology, tissues orcellular material of interest are harvested for isolation of AAVvariants that have successfully infected that target, and the successfulviruses are recovered. Step (c) depicts the Stage 1 enrichment ofsuccessful clones through repeated selection. Step (d) depicts the Stage2 enrichment of selected cap genes which undergo re-diversification andfurther selection steps to iteratively increase viral fitness. Step (e)depicts the variants, identified as hits during Vector Selection Stages1 and 2, which will be manufactured as recombinant AAV vectors andcharacterized for the level of transduction of various cell types andtissue targets. By the nature of the AAV directed evolution process,variants that are disclosed herein have already demonstrated the abilityto transduce retinal cells and deliver a genome (the genome encoding thevariant cap gene) during the selection process.

FIG. 2 provides a retinal flat mount schematic showing where samplesfrom which viral genomes are amplified, are collected across a broadarea of the retina.

FIG. 3 shows a PCR amplification of viral genomes from the ganglion celllayer (GCL), inner nuclear layer (INL), photoreceptor/outer nuclearlayer (ONL), and retinal pigment epithelia (RPE) layer retinal tissuefrom a representative round of selection. Both the right eye (top image)and left eye (bottom image) were injected with library. Inner retina(in), middle retina (mid), and outer/peripheral retina (out) weresampled. Bands within red boxes represent successful amplification ofviral genomes.

FIG. 4A-4D shows frequency of motifs within sequencing analysis. FIG. 4Aprovides Round 3 sequencing analysis. FIG. 4B provides Round 4sequencing analysis. FIG. 4C provides Round 5 sequencing analysis. FIG.4D provides Round 6 sequencing analysis.

FIG. 5 provides a representative three-dimensional model of AAV2containing a random heptamer following amino acid 587.

FIG. 6 provides an alignment of wild-type AAV SEQ ID NOS:1-11 showingamino acid locations between and across the wild-type (naturallyoccurring) serotypes AAV1, AAV2, AAV3A, AAV3B, and AAV4-10.

FIG. 7 provides fundus fluorescence images taken with a HeidelbergSpectralis™ of the retina of an African Green monkey followingintravitreal administration of 2×10¹¹ vector genomes (vg) of AAV2delivering a GFP transgene under the control of the CMV promoter(AAV2.CMV.GFP). Images were taken at baseline (A) and at 14 days (B), 28days (C), and 42 days (D) after injection.

FIG. 8 provides fundus fluorescence images taken with a HeidelbergSpectralis™ the retina of an African Green monkey following intravitrealadministration of 2×10¹¹ vector genomes (vg) of the novel AAV variantLAISDQTKHA+P34A delivering a GFP transgene under the control of the CMVpromoter (LAISDQTKHA+P34A.CMV.GFP). Images taken at baseline (A) and at14 days (B), 28 days (C), and 42 days (D) after injection.

FIG. 9 provides fundus fluorescence images taken with a HeidelbergSpectralis™ of the retinas of Cynomolgus monkeys following intravitrealadministration of the novel AAV variant LAISDQTKHA+P34A delivering a GFPtransgene under the control of the CAG promoter(LAISDQTKHA+P34A.CAG.EGFP). (A) The retina of a monkey injectedintravitreally with 2×10¹¹ vg of vector, imaged 14 days (A1), 21 days(A2), and 28 days (A3) after injection. (B) The retina of a monkeyinjected intravitreally with 1×10¹² vg of vector, imaged 14 days (B1)and 21 days (B2) after injection.

FIGS. 10A-10E provide the results of immunohistochemical analysis of theretina of a monkey injected intravitreally with 1×10¹² vg of the novelAAV variant LAISDQTKHA+P34A delivering a GFP transgene under the controlof the CAG promoter, analyzed three weeks after injection. Allimmunohistochemistry is provided alongside the corresponding fundusfluorescence image, with a red box to denote approximately where in theretina the analysis was performed. FIG. 10A: Robust retinal pigmentepithelium (RPE) and photoreceptor transduction was observed using aGFP-specific antibody (red). Cone photoreceptor immunostaining using anM/L opsin antibody is shown in white. FIGS. 10B and 10C: Robust rod andcone photoreceptor (FIG. 10B) and RPE (FIG. 10C) transduction wasobserved by direct EGFP fluorescence (green) and by immunohistochemistryusing a GFP-specific antibody (red). Melanosomes in RPE appear black inthe image. FIG. 10D: Transduction of cone photoreceptors (identified byM/L opsin, white) and retinal ganglion cells (RGC) in and around thefovea was observed by direct EGFP fluorescence (green) and byimmunohistochemistry using a GFP-specific antibody (red). Images in themiddle panels are a higher magnification (63×) of the area denoted by awhite box in the left panel. FIG. 10E: Transduction of retinal ganglioncells (RGC) and the retinal ganglion cell layer was observed by directEGFP fluorescence (right panels, green; lower right panel is a 63×magnification of the upper right panel); top left panel shows the regionunder brightfield illumination.

FIGS. 11A-11F provides data on the transduction of human retinal pigmentepithelial (RPE) cells in vitro by recombinant AAV virus comprising thenovel AAV variant LAISDQTKHA+P34A capsid and a GFP transgene under thecontrol of the CAG promoter. Cells that were differentiated into RPEcells from a human embryonic stem cell line (FIGS. 11A and 11C) or fromhuman fibroblast-derived induced pluripotent stem cells (FB-iPSC) (FIGS.11B and 11D) were infected with novel AAV variantLAISDQTKHA+P34A.CAG.GFP or wild type control AAV2.CAG.GFP. FIGS. 11A and11B: Immunofluorescence imaging of the cell cultures 7 days afterinfection at an MOI of 500 demonstrates that the novel AAV variantcapsid (left panels) transduces RPE cells better than wild type AAV2capsid (right panels). FIGS. 11C and 11D: Quantification of the percentof GFP-positive RPE cells in each culture by flow cytometry reveals thatthe novel AAV variant capsid provides for a significant, dose-dependentimprovement in the number of cells transduced over wild type AAV2 capsidregardless of cell source. FIGS. 11E and 11F: Quantification of theamount of GFP in each culture by Western blot reveals that the novel AAVvariant provides for significant improvement in expression of thetransgene over wild type capsid regardless of cell source.

DETAILED DESCRIPTION

Before the present methods and compositions are described, it is to beunderstood that this invention is not limited to a particular method orcomposition described and as such may vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

The invention disclosed herein is illustrated in the figures anddescription. However, while particular embodiments are illustrated inthe figures, there is no intention to limit the invention to thespecific embodiment or embodiments illustrated and/or disclosed. Rather,the invention disclosed herein is intended to cover all modifications,alternative constructions, and equivalents falling within the spirit andscope of the invention. As such, the figures are intended to beillustrative and not restrictive.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, some potential andpreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. It is understood that the present disclosuresupersedes any disclosure of an incorporated publication to the extentthere is a contradiction.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

It is noted that as used herein and in the appended claims, the singularforms “a,” “an,” and “the” include plural referents unless the contextclearly dictates otherwise. Thus, for example, reference to “arecombinant AAV virion” includes a plurality of such virions andreference to “the photoreceptor cell” includes reference to one or morephotoreceptor cells and equivalents thereof known to those skilled inthe art and so forth. It is further noted that the claims may be draftedto exclude any optional element. As such, this statement is intended toserve as antecedent basis for use of such exclusive terminology as“solely,” “only” and the like in connection with the recitation of claimelements, or use of a “negative” limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Definitions

Unless otherwise defined, all scientific and technical terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this technology belongs.

Adeno-associated virus is a nonpathogenic parvovirus composed of a 4.7kb single-stranded DNA genome within a non-enveloped, icosahedralcapsid. The genome contains three open reading frames (ORF) flanked byinverted terminal repeats (ITR) that function as the viral origin ofreplication and packaging signal. The rep ORF encodes four nonstructuralproteins that play roles in viral replication, transcriptionalregulation, site-specific integration, and virion assembly. The cap ORFencodes three structural proteins (VP 1-3) that assemble to form a60-mer viral capsid. Finally, an ORF present as an alternate readingframe within the cap gene produces the assembly-activating protein(AAP), a viral protein that localizes AAV capsid proteins to thenucleolus and functions in the capsid assembly process.

There are several naturally occurring (“wild-type”) serotypes and over100 known variants of AAV, each of which differs in amino acid sequence,particularly within the hypervariable regions of the capsid proteins,and thus in their gene delivery properties. No AAV has been associatedwith any human disease, making recombinant AAV attractive for clinicalapplications.

For the purposes of the disclosure herein, the terminology “AAV” is anabbreviation for adeno-associated virus, including, without limitation,the virus itself and derivatives thereof. Except where otherwiseindicated, the terminology refers to all subtypes or serotypes and bothreplication-competent and recombinant forms. The term “AAV” includes,without limitation, AAV type 1 (AAV-1 or AAV1), AAV type 2 (AAV-2 orAAV2), AAV type 3A (AAV-3A or AAV3A), AAV type 3B (AAV-3B or AAV3B), AAVtype 4 (AAV-4 or AAV4), AAV type 5 (AAV-5 or AAV5), AAV type 6 (AAV-6 orAAV6), AAV type 7 (AAV-7 or AAV7), AAV type 8 (AAV-8 or AAV8), AAV type9 (AAV-9 or AAV9), AAV type 10 (AAV-10 or AAV10 or AAVrh10), avian AAV,bovine AAV, canine AAV, caprine AAV, equine AAV, primate AAV,non-primate AAV, and ovine AAV. “Primate AAV” refers to AAV that infectprimates, “non-primate AAV” refers to AAV that infect non-primatemammals, “bovine AAV” refers to AAV that infect bovine mammals, etc.

The genomic sequences of various serotypes of AAV, as well as thesequences of the native terminal repeats (TRs), Rep proteins, and capsidsubunits are known in the art. Such sequences may be found in theliterature or in public databases such as GenBank. See, e.g., GenBankAccession Numbers NC_002077.1 (AAV1), AF063497.1 (AAV1), NC_001401.2(AAV2), AF043303.1 (AAV2), J01901.1 (AAV2), U48704.1 (AAV3A),NC_001729.1 (AAV3A), AF028705.1 (AAV3B), NC_001829.1 (AAV4), U89790.1(AAV4), NC_006152.1 (AA5), AF085716.1 (AAV-5), AF028704.1 (AAV6),NC_006260.1 (AAV7), AF513851.1 (AAV7), AF513852.1 (AAV8) NC_006261.1(AAV-8), AY530579.1 (AAV9), AAT46337 (AAV10) and AAO88208 (AAVrh10); thedisclosures of which are incorporated by reference herein for teachingAAV nucleic acid and amino acid sequences. See also, e.g., Srivistava etal. (1983) J. Virology 45:555; Chiorini et al. (1998) J. Virology71:6823; Chiorini et al. (1999) J. Virology 73: 1309; Bantel-Schaal etal. (1999) J. Virology 73:939; Xiao et al. (1999) J. Virology 73:3994;Muramatsu et al. (1996) Virology 221:208; Shade et. al. (1986) J. Virol.58:921; Gao et al. (2002) Proc. Nat. Acad. Sci. USA 99: 11854; Moris etal. (2004) Virology 33:375-383; international patent publications WO00/28061, WO 99/61601, WO 98/11244; and U.S. Pat. No. 6,156,303.

The sequences of naturally existing cap (capsid) proteins associatedwith AAV serotypes are known in the art and include those disclosedherein as AAV1 (SEQ ID NO:1), AAV2 (SEQ ID NO:2), AAV3A (SEQ ID NO:3),AAV3B (SEQ ID NO:4), AAV4 (SEQ ID NO:5), AAV5 (SEQ ID NO:6), AAV6 (SEQID NO:7), AAV7 (SEQ ID NO:8), AAV8 (SEQ ID NO:9), AAV9 (SEQ ID NO:10),AAV10 (SEQ ID NO:1), and AAVrh10 (SEQ ID NO:12). The terms “variant AAVcapsid protein” or “AAV variant’ refer to an AAV capsid proteincomprising an amino acid sequence that includes at least onemodification or substitution (including deletion, insertion, pointmutation, etc.) relative to a naturally existing or “wild-type” AAVcapsid protein sequences, e.g. as set forth in SEQ ID NO:1-12 herein. Avariant AAV capsid protein may have about 80% identity or more to theamino acid sequence of a wild type capsid protein, for example, 85%identity or more, 90% identity or more, or 95% identity or more to theamino acid sequence of the wild type capsid protein, for example, 98% or99% identity to the wild type capsid protein. A variant AAV capsidprotein may not be a wild type capsid protein.

For the purposes of the disclosure herein, “AAV virion” or “AAV viralparticle” refers to a viral particle composed of at least one AAV capsidprotein and an encapsidated AAV polynucleotide.

For the purposes of the disclosure herein, the terminology “rAAV” is anabbreviation that refers to recombinant adeno-associated virus.“Recombinant,” as applied to a polynucleotide means that thepolynucleotide is the product of various combinations of cloning,restriction or ligation steps, and other procedures that result in aconstruct that is distinct from a polynucleotide found in nature. Arecombinant virus is a viral particle comprising a recombinantpolynucleotide. The terms respectively include replicates of theoriginal polynucleotide construct and progeny of the original virusconstruct.

The term “rAAV vector” encompasses rAAV virions (i.e., rAAV viralparticles) (e.g., an infectious rAAV virion), which by definitioninclude an rAAV polynucleotide; and also encompasses polynucleotidesencoding rAAV (e.g., a single stranded polynucleotide encoding rAAV(ss-rAAV); a double stranded polynucleotide encoding rAAV (ds-rAAV),e.g., plasmids encoding rAAV; and the like).

If an AAV virion comprises a heterologous polynucleotide (i.e. apolynucleotide other than a wild-type AAV genome, e.g., a transgene tobe delivered to a target cell, an RNAi agent or CRISPR agent to bedelivered to a target cell, etc.), it is typically referred to as a“recombinant AAV (rAAV) virion” or an “rAAV viral particle.” In general,the heterologous polynucleotide is flanked by at least one, andgenerally by two, AAV inverted terminal repeat sequences (ITRs).

The term “packaging” refers to a series of intracellular events thatresult in the assembly and encapsidation of an AAV particle. AAV “rep”and “cap” genes refer to polynucleotide sequences encoding replicationand encapsidation proteins of adeno-associated virus. AAV rep and capare referred to herein as AAV “packaging genes.”

The terminology “helper virus” for AAV refers to a virus that allows AAV(e.g. wild-type AAV) to be replicated and packaged by a mammalian cell.A variety of such helper viruses for AAV are known in the art, includingadenoviruses, herpesviruses and poxviruses such as vaccinia. Theadenoviruses encompass a number of different subgroups, althoughAdenovirus type 5 of subgroup C is most commonly used. Numerousadenoviruses of human, non-human mammalian and avian origin are knownand available from depositories such as the ATCC. Viruses of the herpesfamily include, for example, herpes simplex viruses (HSV) andEpstein-Barr viruses (EBV), as well as cytomegaloviruses (CMV) andpseudorabies viruses (PRV); which are also available from depositoriessuch as ATCC.

The terminology “helper virus function(s)” refers to function(s) encodedin a helper virus genome which allow AAV replication and packaging (inconjunction with other requirements for replication and packagingdescribed herein). As described herein, “helper virus function” may beprovided in a number of ways, including by providing helper virus orproviding, for example, polynucleotide sequences encoding the requisitefunction(s) to a producer cell in trans. For example, a plasmid or otherexpression vector comprising nucleotide sequences encoding one or moreadenoviral proteins is transfected into a producer cell along with anrAAV vector.

The terminology “infectious” virus or viral particle is one thatcomprises a competently assembled viral capsid and is capable ofdelivering a polynucleotide component into a cell for which the viralspecies is tropic. The term does not necessarily imply any replicationcapacity of the virus. Assays for counting infectious viral particlesare described elsewhere in this disclosure and in the art. Viralinfectivity can be expressed as the ratio of infectious viral particlesto total viral particles. Methods of determining the ratio of infectiousviral particle to total viral particle are known in the art. See, e.g.,Grainger et al. (2005) Mol. Ther. 11: S337 (describing a TCID50infectious titer assay); and Zolotukhin et al. (1999) Gene Ther. 6:973.See also the Examples.

The term “tropism” as used herein refers to the preferential targetingby a virus (e.g., an AAV) of cells of a particular host species or ofparticular cell types within a host species. For example, a virus thatcan infect cells of the heart, lung, liver, and muscle has a broader(i.e., increased) tropism relative to a virus that can infect only lungand muscle cells. Tropism can also include the dependence of a virus onparticular types of cell surface molecules of the host. For example,some viruses can infect only cells with surface glycosaminoglycans,while other viruses can infect only cells with sialic acid (suchdependencies can be tested using various cells lines deficient inparticular classes of molecules as potential host cells for viralinfection). In some cases, the tropism of a virus describes the virus'srelative preferences. For example, a first virus may be able to infectall cell types but is much more successful in infecting those cells withsurface glycosaminoglycans. A second virus can be considered to have asimilar (or identical) tropism as the first virus if the second virusalso prefers the same characteristics (e.g., the second virus is alsomore successful in infecting those cells with surfaceglycosaminoglycans), even if the absolute transduction efficiencies arenot similar. For example, the second virus might be more efficient thanthe first virus at infecting every given cell type tested, but if therelative preferences are similar (or identical), the second virus canstill be considered to have a similar (or identical) tropism as thefirst virus. In some embodiments, the tropism of a virion comprising asubject variant AAV capsid protein is not altered relative to anaturally occurring virion. In some embodiments, the tropism of a virioncomprising a subject variant AAV capsid protein is expanded (i.e.,broadened) relative to a naturally occurring virion. In someembodiments, the tropism of a virion comprising a subject variant AAVcapsid protein is reduced relative to a naturally occurring virion.

The terminology “replication-competent” virus (e.g. areplication-competent AAV) refers to a phenotypically wild-type virusthat is infectious, and is also capable of being replicated in aninfected cell (i.e. in the presence of a helper virus or helper virusfunctions). In the case of AAV, replication competence generallyrequires the presence of functional AAV packaging genes. In general,rAAV vectors as described herein are replication-incompetent inmammalian cells (especially in human cells) by virtue of the lack of oneor more AAV packaging genes. Typically, such rAAV vectors lack any AAVpackaging gene sequences in order to minimize the possibility thatreplication competent AAV are generated by recombination between AAVpackaging genes and an incoming rAAV vector. In many embodiments, rAAVvector preparations as described herein are those which contain few ifany replication competent AAV (rcAAV, also referred to as RCA) (e.g.,less than about 1 rcAAV per 10² rAAV particles, less than about 1 rcAAVper 10⁴ rAAV particles, less than about 1 rcAAV per 10 rAAV particles,less than about 1 rcAAV per 10¹² rAAV particles, or no rcAAV).

The term “polynucleotide” refers to a polymeric form of nucleotides ofany length, including deoxyribonucleotides or ribonucleotides, oranalogs thereof. A polynucleotide may comprise modified nucleotides,such as methylated nucleotides and nucleotide analogs, and may beinterrupted by non-nucleotide components. If present, modifications tothe nucleotide structure may be imparted before or after assembly of thepolymer. The term polynucleotide, as used herein, refers interchangeablyto double- and single-stranded molecules. Unless otherwise specified orrequired, any embodiment herein that comprises a polynucleotideencompasses both the double-stranded form and each of two complementarysingle-stranded forms known or predicted to make up the double-strandedform.

A polynucleotide or polypeptide has a certain percent “sequenceidentity” to another polynucleotide or polypeptide, meaning that, whenaligned, that percentage of bases or amino acids are the same whencomparing the two sequences. Sequence similarity can be determined in anumber of different manners. To determine sequence identity, sequencescan be aligned using the methods and computer programs, including BLAST,available over the world wide web at ncbi.nlm.nih.gov/BLAST/. Anotheralignment algorithm is FASTA, available in the Genetics Computing Group(GCG) package, from Madison, Wis., USA, a wholly owned subsidiary ofOxford Molecular Group, Inc. Other techniques for alignment aredescribed in Methods in Enzymology, vol. 266: Computer Methods forMacromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press,Inc., a division of Harcourt Brace & Co., San Diego, Calif., USA. Ofparticular interest are alignment programs that permit gaps in thesequence. The Smith-Waterman is one type of algorithm that permits gapsin sequence alignments. See Meth. Mol. Biol. 70: 173-187 (1997). Also,the GAP program using the Needleman and Wunsch alignment method can beutilized to align sequences. See J. Mol. Biol. 48: 443-453 (1970).

The term “gene” refers to a polynucleotide that performs a function ofsome kind in the cell. For example, a gene can contain an open readingframe that is capable of encoding a gene product. One example of a geneproduct is a protein, which is transcribed and translated from the gene.Another example of a gene product is an RNA, e.g. a functional RNAproduct, e.g., an aptamer, an interfering RNA, a ribosomal RNA (rRNA), atransfer RNA (tRNA), a non-coding RNA (ncRNA), a guide RNA fornucleases, etc., which is transcribed but not translated.

The terminology “gene expression product” or “gene product” is amolecule resulting from expression of a particular gene, as definedabove. Gene expression products include, e.g., a polypeptide, anaptamer, an interfering RNA, a messenger RNA (mRNA), an rRNA, a tRNA, anon-coding RNA (ncRNA), and the like.

The term “siRNA agent” (“small interfering” or “short interfering RNA”(or siRNA)) is an RNA duplex of nucleotides that is targeted to a geneinterest (a “target gene”). An “RNA duplex” refers to the structureformed by the complementary pairing between two regions of a RNAmolecule, forming a region of double stranded RNA (dsRNA). siRNA is“targeted” to a gene in that the nucleotide sequence of the duplexportion of the siRNA is complementary to a nucleotide sequence of thetargeted gene. In some embodiments, the length of the duplex of siRNAsis less than 30 nucleotides. In some embodiments, the duplex can be 29,28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11or 10 nucleotides in length. In some embodiments, the length of theduplex is 19-25 nucleotides in length. In some embodiments,siRNA-mediated gene targeting is accomplished through the use ofDNA-directed RNA interference (ddRNAi) which is a gene-silencingtechnique that utilizes DNA constructs to activate an animal cell'sendogenous RNA interference (RNAi) pathways. Such DNA constructs aredesigned to express self-complementary double-stranded RNAs, typicallyshort-hairpin RNAs (shRNA), that once processed bring about silencing ofa target gene or genes. Any RNA, including endogenous mRNAs or viralRNAs, can be silenced by designing constructs to express double-strandedRNA complementary to the desired mRNA target. As such, the RNA duplexportion of an siRNA agent can be part of a short hairpin structurereferred to as shRNA. In addition to the duplex portion, the hairpinstructure may contain a loop portion positioned between the twosequences that form the duplex. The loop can vary in length. In someembodiments the loop is 5, 6, 7, 8, 9, 10, 11, 12 or 13 nucleotides inlength. The hairpin structure can also contain 3′ or 5′ overhangportions. In some embodiments, the overhang is a 3′ or a 5′ overhang 0,1, 2, 3, 4 or 5 nucleotides in length. In general, the level ofexpression product (e.g., mRNA, polypeptide, etc.) of a target gene isreduced by an siRNA agent (e.g., an siRNA, an shRNA, etc.) that containsspecific double stranded nucleotide sequences that are complementary toat least a 19-25 nucleotide long segment (e.g., a 20-21 nucleotidesequence) of the target gene transcript, including the 5′ untranslated(UT) region, the ORF, or the 3′ UT region. In some embodiments, shortinterfering RNAs are about 19-25 nt in length. See, e.g., PCTapplications WOO/44895, WO99/32619, WO01/75164, WO01/92513, WO01/29058,WO01/89304, WO02/16620, and WO02/29858; and U.S. Patent Publication No.20040023390 for descriptions of siRNA technology. The siRNA and/or shRNAcan be encoded by a nucleic acid sequence, and the nucleic acid sequencecan also include a promoter. The nucleic acid sequence can also includea polyadenylation signal. In some embodiments, the polyadenylationsignal is a synthetic minimal polyadenylation signal.

The terminology “antisense RNA” encompasses RNA that is complementary toa gene expression product. For example, an antisense RNA targeted to aspecific mRNA is an RNA-based agent (or can be a modified RNA) that iscomplementary to the mRNA, where hybridization of the antisense RNA tothe mRNA alters the expression of the mRNA (e.g., via altering thestability of the RNA, altering the translation of the RNA, etc.). Alsoincluded in “antisense RNA” are nucleic acids encoding an antisense RNA.

With regards to “CRISPR/Cas9 agents”, the term “CRISPR” encompassesClustered regularly interspaced short palindromicrepeats/CRISPR-associated (Cas) systems that evolved to provide bacteriaand archaea with adaptive immunity against viruses and plasmids by usingCRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids.The Cas9 protein (or functional equivalent and/or variant thereof, i.e.,Cas9-like protein) naturally contains DNA endonuclease activity thatdepends on association of the protein with two naturally occurring orsynthetic RNA molecules called crRNA and tracrRNA (also called guideRNAs). In some cases, the two molecules are covalently linked to form asingle molecule (also called a single guide RNA (“sgRNA”)). Thus, theCas9 or Cas9-like protein associates with a DNA-targeting RNA (whichterm encompasses both the two-molecule guide RNA configuration and thesingle-molecule guide RNA configuration), which activates the Cas9 orCas9-like protein and guides the protein to a target nucleic acidsequence.

If the Cas9 or Cas9-like protein retains its natural enzymatic function,it will cleave target DNA to create a double-strand break, which canlead to genome alteration (i.e., editing: deletion, insertion (when adonor polynucleotide is present), replacement, etc.), thereby alteringgene expression. Some variants of Cas9 (which variants are encompassedby the term Cas9-like) have been altered such that they have a decreasedDNA cleaving activity (in some cases, they cleave a single strandinstead of both strands of the target DNA, while in other cases, theyhave severely reduced to no DNA cleavage activity). Cas9-like proteinswith decreased DNA-cleavage activity (even no DNA-cleaving activity) canstill be guided to a target DNA to block RNA polymerase activity.Alternatively, the Cas9 or Cas9-like protein may be modified by fusing aVP64 transcription activation domain to the Cas9 protein andcodelivering the fusion protein with a MS2-P65-HSF1 helper protein and asingle guide RNA comprising MS2 RNA aptamers at the tetraloop andstem-loop to form a Synergistic Activation Mediator (Cas9-SAM) complexin the cell that activates transcription. Thus enzymatically inactiveCas9-like proteins can be targeted to a specific location in a targetDNA by a DNA-targeting RNA in order to block or activate transcriptionof the target DNA. The term “CRISPR/Cas9 agents” as used hereinencompasses all forms of CRISPR/Cas9 as described above or as known inthe art.

Detailed information regarding CRISPR agents can be found, for examplein (a) Jinek et. al., Science. 2012 Aug. 17; 337(6096):816-21: “Aprogrammable dual-RNA-guided DNA endonuclease in adaptive bacterialimmunity”; (b) Qi et al., Cell. 2013 Feb. 28; 152(5): 1173-83:“Repurposing CRISPR as an RNA-guided platform for sequence-specificcontrol of gene expression”, and (c) U.S. patent application Ser. No.13/842,859 and PCT application number PCT/US13/32589; all of which arehereby incorporated by reference in their entirety. Thus, the term“CRISPR agent” as used herein encompasses any agent (or nucleic acidencoding such an agent), comprising naturally occurring and/or syntheticsequences, that can be used in the Cas9-based system (e.g., a Cas9 orCas9-like protein; any component of a DNA-targeting RNA, e.g., acrRNA-like RNA, a tracrRNA-like RNA, a single guide RNA, etc.; a donorpolynucleotide; and the like).

By “Zinc-finger nucleases” (ZFNs) it is meant artificial DNAendonucleases generated by fusing a zinc finger DNA binding domain to aDNA cleavage domain. ZFNs can be engineered to target desired DNAsequences and this enables zinc-finger nucleases to cleave unique targetsequences. When introduced into a cell, ZFNs can be used to edit targetDNA in the cell (e.g., the cell's genome) by inducing double strandbreaks. For more information on the use of ZFNs, see, for example: Asuriet al., Mol Ther. 2012 February; 20(2):329-38; Bibikova et al. Science.2003 May 2; 300(5620):764; Wood et al. Science. 2011 Jul. 15;333(6040):307; Ochiai et al. Genes Cells. 2010 August; 15(8):875-85;Takasu et. al., Insect Biochem Mol Biol. 2010 October; 40(10):759-65;Ekker et al, Zebrafish 2008 Summer, 5(2): 121-3; Young et al, Proc NatlAcad Sci USA. 2011 Apr. 26; 108(17):7052-7; Goldberg et al, Cell. 2010Mar. 5; 140(5):678-91; Geurts et al, Science. 2009 Jul. 24;325(5939):433; Flisikowska et al, PLoS One. 2011; 6(6):e21045. doi:10.1371/journal.pone.0021045. Epub 2011 Jun. 13; Hauschild et al, ProcNat Acad Sci USA. 2011 Jul. 19; 108(29): 12013-7; and Yu et al, CellRes. 2011 November; 21(11): 1638-40; all of which are hereinincorporated by reference for their teachings related to ZFNs. The term“ZFN agent” encompasses a zinc finger nuclease and/or a polynucleotidecomprising a nucleotide sequence encoding a zinc finger nuclease.

The terminology “Transcription activator-like effector nuclease” or“TALEN” agents refers to Transcription activator-like effector nucleases(TALENs) are artificial DNA endonucleases generated by fusing a TAL(Transcription activator-like) effector DNA binding domain to a DNAcleavage domain. TALENS can be quickly engineered to bind practicallyany desired DNA sequence and when introduced into a cell, TALENs can beused to edit target DNA in the cell (e.g., the cell's genome) byinducing double strand breaks. For more information on the use ofTALENs, see, for example: Hockemeyer et al. Nat Biotechnol. 2011 Jul. 7;29(8):731-4; Wood et al. Science. 2011 Jul. 15; 333(6040):307; Tesson etal. Nat Biotechnol. 2011 Aug. 5; 29(8):695-6; and Huang et. al., NatBiotechnol. 2011 Aug. 5; 29(8):699-700; all of which are hereinincorporated by reference for their teachings related to TALENs. Theterm “TALEN agent” encompasses a TALEN and/or a polynucleotidecomprising a nucleotide sequence encoding a TALEN.

The terminology “control element” or “control sequence” refers to anucleotide sequence involved in an interaction of molecules thatcontributes to the functional regulation of a polynucleotide, includingreplication, duplication, transcription, splicing, translation, ordegradation of the polynucleotide. The regulation may affect thefrequency, speed, or specificity of the process, and may be enhancing orinhibitory in nature. Control elements known in the art include, forexample, transcriptional regulatory sequences such as promoters andenhancers. A promoter is a DNA region capable under certain conditionsof binding RNA polymerase and initiating transcription of a codingregion usually located downstream (in the 3′ direction) from thepromoter. Promoters may be ubiquitously acting, i.e. active in many celltypes, e.g. CAG or CMV promoters; or tissue or cell specific, e.g. therho promoter, which is active in rods, or the opsin promoter, which isactive in cones.

The terminology “operatively linked” or “operably linked” refers to ajuxtaposition of genetic elements, wherein the elements are in arelationship permitting them to operate in the expected manner. Forinstance, a promoter is operatively linked to a coding region if thepromoter helps initiate transcription of the coding sequence. There maybe intervening residues between the promoter and coding region so longas this functional relationship is maintained.

The terminology “expression vector” encompasses a vector comprising apolynucleotide region which encodes a polypeptide of interest, and isused for effecting the expression of the protein in an intended targetcell. An expression vector may also comprise control elementsoperatively linked to the encoding region to facilitate expression ofthe protein in the target. The combination of control elements and agene or genes to which they are operably linked for expression issometimes referred to as an “expression cassette,” a large number ofwhich are known and available in the art or can be readily constructedfrom components that are available in the art.

The term “heterologous” means derived from a genotypically distinctentity from that of the rest of the entity to which it is beingcompared. For example, a polynucleotide introduced by geneticengineering techniques into a plasmid or vector derived from a differentspecies is a heterologous polynucleotide. A promoter removed from itsnative coding sequence and operatively linked to a coding sequence withwhich it is not naturally found linked is a heterologous promoter. Thus,for example, an rAAV that includes a heterologous nucleic acid sequenceencoding a heterologous gene product is an rAAV that includes apolynucleotide not normally included in a naturally-occurring, wild-typeAAV, and the encoded heterologous gene product is a gene product notnormally encoded by a naturally-occurring, wild type AAV.

The terminology “genetic alteration” and “genetic modification” (andgrammatical variants thereof), are used interchangeably herein to referto a process wherein a genetic element (e.g., a polynucleotide) isintroduced into a cell other than by mitosis or meiosis. The element maybe heterologous to the cell, or it may be an additional copy or improvedversion of an element already present in the cell. Genetic alterationmay be effected, for example, by transfecting a cell with a recombinantplasmid or other polynucleotide through any process known in the art,such as electroporation, calcium phosphate precipitation, or contactingwith a polynucleotide-liposome complex. Genetic alteration may also beeffected, for example, by transduction or infection with a DNA or RNAvirus or viral vector. Generally, the genetic element is introduced intoa chromosome or mini-chromosome in the cell; but any alteration thatchanges the phenotype and/or genotype of the cell and its progeny isincluded in this term.

With regards to cell modification, the terminology “geneticallymodified” or “transformed” or “transfected” or “transduced” by exogenousDNA (e.g. via a recombinant virus) refers to when such DNA has beenintroduced inside the cell. The presence of the exogenous DNA results inpermanent or transient genetic change. The transforming DNA may or maynot be integrated (covalently linked) into the genome of the cell. A“clone” is a population of cells derived from a single cell or commonancestor by mitosis. A “cell line” is a clone of a primary cell that iscapable of stable growth in vitro for many generations.

As used herein, a cell is said to be “stably” altered, transduced,genetically modified, or transformed with a genetic sequence if thesequence is available to perform its function during extended culture ofthe cell in vitro and/or for an extended period of time in vivo.Generally, such a cell is “heritably” altered (genetically modified) inthat a genetic alteration is introduced which is also inheritable byprogeny of the altered cell.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength. The terms also encompass an amino acid polymer that has beenmodified; for example, disulfide bond formation, glycosylation,lipidation, phosphorylation, or conjugation with a labeling component.Polypeptides such as anti-angiogenic polypeptides, neuroprotectivepolypeptides, and the like, when discussed in the context of deliveringa gene product to a mammalian subject, and compositions therefor, referto the respective intact polypeptide, or any fragment or geneticallyengineered derivative thereof which retains the desired biochemicalfunction of the intact protein. Similarly, references to nucleic acidsencoding anti-angiogenic polypeptides, nucleic acids encodingneuroprotective polypeptides, and other such nucleic acids for use indelivery of a gene product to a mammalian subject (which may be referredto as “transgenes” to be delivered to a recipient cell), includepolynucleotides encoding the intact polypeptide or any fragment orgenetically engineered derivative possessing the desired biochemicalfunction.

As used herein, an “isolated” plasmid, nucleic acid, vector, virus,virion, host cell, protein, or other substance refers to a preparationof the substance devoid of at least some of the other components thatmay also be present where the substance or a similar substance naturallyoccurs or is initially prepared from. Thus, for example, an isolatedsubstance may be prepared by using a purification technique to enrich itfrom a source mixture. Enrichment can be measured on an absolute basis,such as weight per volume of solution, or it can be measured in relationto a second, potentially interfering substance present in the sourcemixture. Increasing enrichments of the embodiments of this disclosureare increasingly more isolated. An isolated plasmid, nucleic acid,vector, virus, host cell, or other substance is in some embodimentspurified, e.g., from about 8% to about 90% pure, at least about 90%pure, at least about 95% pure, at least about 98% pure, or at leastabout 99%, or more, pure.

As used herein, the terms “treatment,” “treating,” and the like, referto obtaining a desired pharmacologic and/or physiologic effect. Theeffect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of a partial or complete cure for a disease and/or adverse effectattributable to the disease. “Treatment,” as used herein, covers anytreatment of a disease in a mammal, particularly in a human, andincludes: (a) preventing the disease (and/or symptoms caused by thedisease) from occurring in a subject which may be predisposed to thedisease or at risk of acquiring the disease but has not yet beendiagnosed as having it; (b) inhibiting the disease (and/or symptomscaused by the disease), i.e., arresting its development; and (c)relieving the disease (and/or symptoms caused by the disease), i.e.,causing regression of the disease (and/or symptoms caused by thedisease), i.e., ameliorating the disease and/or one or more symptoms ofthe disease. For example, the subject compositions and methods may bedirected towards the treatment of retinal disease. Nonlimiting methodsfor assessing retinal diseases and the treatment thereof includemeasuring retinal function and changes thereof, e.g. changes in visualacuity (e.g. best-corrected visual acuity [BCVA], ambulation,navigation, object detection and discrimination), changes in visualfield (e.g. static and kinetic visual field perimetry), clinicalexamination (e.g. slit lamp examination of the anterior and posteriorsegments of the eye), electrophysiological responsiveness to allwavelengths of light and dark (e.g. all forms of electroretinography(ERG) [full-field, multifocal and pattern], all forms of visual evokedpotential (VEP), electrooculography (EOG), color vision, dark adaptationand/or contrast sensitivity; measuring changes in anatomy or healthusing anatomical and/or photographic measures, e.g. Optical ConherenceTomography (OCT), fundus photography, adaptive optics scanning laserophthalmoscopy, fluorescence and/or autofluorescence; measuring ocularmotility and eye movements (e.g. nystagmus, fixation preference, andstability), measuring reported outcomes (patient-reported changes invisual and non-visually-guided behaviors and activities,patient-reported outcomes [PRO], questionnaire-based assessments ofquality-of-life, daily activities and measures of neurological function(e.g. functional Magnetic Resonance Imaging (MRI)).

The terms “individual,” “host,” “subject,” and “patient” are usedinterchangeably herein, and refer to a mammal, including, but notlimited to, humans; non-human primates, including simians; mammaliansport animals (e.g., horses); mammalian farm animals (e.g., sheep,goats, etc.); mammalian pets (dogs, cats, etc.); and rodents (e.g.,mice, rats, etc.).

In some embodiments, the individual is a human who has previously beennaturally exposed to AAV and as a result harbors anti-AAV antibodies(i.e., AAV neutralizing antibodies). In some embodiments, the individualis a human who has previously been administered an AAV vector (and as aresult may harbor anti-AAV antibodies) and needs re-administration ofvector for treatment of a different condition or for further treatmentof the same condition. Based on positive results in clinical trialsinvolving AAV gene delivery to, for example, liver, muscle, andretina—all tissues affected by neutralizing antibodies against thisvehicle—there are many such therapeutic applications/disease targets.

The term “effective amount” as used herein is an amount sufficient toeffect beneficial or desired clinical results. An effective amount canbe administered in one or more administrations. For purposes of thisdisclosure, an effective amount of a compound (e.g., an infectious rAAVvirion) is an amount that is sufficient to palliate, ameliorate,stabilize, reverse, prevent, slow or delay the progression of (and/orsymptoms associated with) a particular disease state (e.g., a retinaldisease). Accordingly, an effective amount of an infectious rAAV virionis an amount of the infectious rAAV virion that is able to effectivelydeliver a heterologous nucleic acid to a target cell (or target cells)of the individual. Effective amounts may be determined preclinically by,e.g., detecting in the cell or tissue the gene product (RNA, protein)that is encoded by the heterologous nucleic acid sequence usingtechniques that are well understood in the art, e.g. RT-PCR, westernblotting, ELISA, fluorescence or other reporter readouts, and the like.Effective amounts may be determined clinically by, e.g. detecting achange in the onset or progression of disease using methods known in theart, e.g. fundus autofluorescence, fluorescein angiography, OCT,microperimetry, adaptive optics, etc. and the like, as described hereinand as known in the art.

The terminology “retinal cell” refers herein to any of the cell typesthat comprise the retina, such as, without limitation, retinal ganglion(RG) cells, amacrine cells, horizontal cells, bipolar cells,photoreceptor cells, Müller glial cells, microglial cells, and retinalpigmented epithelium (RPE). The terminology “photoreceptor cells” refersherein to, without limitation, rod cells or “rods” and cone cells or“cones”. The terminology “Müller cells” or “Müller glia” refers to glialcells that support neurons in the vertebrate retina.

The terminology “directed evolution” refers to a capsid engineeringmethodology, in vitro and/or in vivo, which emulates natural evolutionthrough iterative rounds of genetic diversification and selectionprocesses, thereby accumulating beneficial mutations that progressivelyimprove the function of a biomolecule. Directed evolution often involvesan in vivo method referred to as “biopanning” for selection of AAVvariants from a library which variants possess a more efficient level ofinfectivity of a cell or tissue type of interest.

DETAILED DESCRIPTION

Adeno-associated viruses (AAVs) are a family of parvoviruses with a 4.7kb single-stranded DNA genome contained inside a non-enveloped capsid.The viral genome of a naturally occurring AAV has 2 inverted terminalrepeats (ITR)—which function as the viral origin of replication andpackaging signal—flanking 2 primary open reading frames (ORF): rep(encoding proteins that function in viral replication, transcriptionalregulation, site-specific integration, and virion assembly) and cap. Thecap ORF codes for 3 structural proteins that assemble to form a 60-merviral capsid. Many naturally occurring AAV variants and serotypes havebeen isolated, and none have been associated with human disease.

Recombinant versions of AAV can be used as gene delivery vectors, wherea marker or therapeutic gene of interest is inserted between the ITRs inplace of rep and cap. These vectors have been shown to transduce bothdividing and non-dividing cells in vitro and in vivo and can result instable transgene expression for years in post-mitotic tissue. See e.g.,Knipe D M, Howley P M. Fields' Virology. Lippincott Williams & Wilkins,Philadelphia, Pa., USA, 2007; Gao G-P, Alvira M R, Wang L, Calcedo R,Johnston J, Wilson J M. Novel adeno-associated viruses from rhesusmonkeys as vectors for human gene therapy. Proc Natl Acad Sci USA 2002;99: 11854-9; Atchison R W, Casto B C, Hammon W M. Adenovirus-AssociatedDefective Virus Particles. Science 1965; 149: 754-6; Hoggan M D,Blacklow N R, Rowe W P. Studies of small DNA viruses found in variousadenovirus preparations: physical, biological, and immunologicalcharacteristics. Proc Nal Acad Sci USA 1966; 55: 1467-74; Blacklow N R,Hoggan M D, Rowe W P. Isolation of adenovirus-associated viruses fromman. Proc Natl Acad Sci USA 1967; 58: 1410-5; Bantel-Schaal U, zurHausen H. Characterization of the DNA of a defective human parvovirusisolated from a genital site. Virology 1984; 134: 52-63; Mayor H D,Melnick J L. Small deoxyribonucleic acid-containing viruses(picodnavirus group). Nature 1966; 210: 331-2; Mori S, Wang L, TakeuchiT, Kanda T. Two novel adeno-associated viruses from cynomolgus monkey:pseudotyping characterization of capsid protein. Virology 2004; 330:375-83; Flotte T R. Gene therapy progress and prospects: recombinantadeno-associated virus (rAAV) vectors. Gene Ther 2004; 11: 805-10.

Recombinant AAV (referred to herein simply as “AAV”) has yieldedpromising results in an increasing number of clinical trials. However,there are impediments to gene delivery that may limit AAV's utility,such as anti-capsid immune responses, low transduction of certaintissues, an inability for targeted delivery to specific cell types and arelatively low carrying capacity. In many situations, there isinsufficient mechanistic knowledge to effectively empower rationaldesign with the capacity to improve AAV. As an alternative, directedevolution has emerged as a strategy to create novel AAV variants thatmeet specific biomedical needs. Directed evolution strategies harnessgenetic diversification and selection processes to enable theaccumulation of beneficial mutations that progressively improve thefunction of a biomolecule. In this process, wild-type AAV cap genes arediversified by several approaches to create large genetic libraries thatare packaged to generate libraries of viral particles, and selectivepressure is then applied to isolate novel variants that can overcomegene delivery barriers. Importantly, the mechanistic basis underlying agene delivery problem does not need to be known for directed evolutionof function, which can thus accelerate the development of enhancedvectors.

Typically, the variants disclosed herein were generated through use ofan AAV library and/or libraries. Such an AAV library or libraries is/aregenerated by mutating the cap gene, the gene which encodes thestructural proteins of the AAV capsid, by a range of directed evolutiontechniques known by and readily available to the skilled artisan in thefield of viral genome engineering. See e.g., Bartel et al. Am. Soc. GeneCell Ther. 15^(th) Annu. Meet. 20, S140 (2012); Bowles, D. et al. J.Virol. 77, 423-432 (2003); Gray et al. Mol. Ther. 18, 570-578 (2010);Grimm, D. et al. J. Virol. 82, 5887-5911; Koerber, J. T. et al. Mol.Ther. 16, 1703-1709 (2008); Li W. et al. Mol. Ther. 16, 1252-1260(2008); Koerber, J. T. et al. Methods Mol. Biol. 434, 161-170 (2008);Koerber, J. T. et al. Hum. Gene Ther. 18, 367-378 (2007); and Koerber,J. T. et al. Mol. Ther. 17, 2088-2095 (2009). Such techniques, withoutlimitation, are as follows: i) Error-prone PCR to introduce random pointmutations into the AAV cap open reading frame (ORF) at a predetermined,modifiable rate; ii) In vitro or in vivo viral recombination or “DNAshuffling” to generate random chimeras of AAV cap genes to yield a genelibrary with multiple AAV serotypes; iii) Random peptide insertions atdefined sites of the capsid by ligation of degenerate oligonucleotidesin the cap ORF; iv) Defined insertions of peptide-encoding sequencesinto random locations of the AAV cap ORF using transposon mutagenesis;v) Replacing surface loops of AAV capsids with libraries of peptidesequences bioinformationally designed based on the level of conservationof each amino acid position among natural AAV serotypes and variants togenerate “loop-swap” libraries; vi) Random amino acid substitution atpositions of degeneracy between AAV serotypes to generate libraries ofancestral variants (Santiago-Ortiz et al., 2015); and a combination ofsuch techniques thereof.

DNA shuffling generates chimeras which combine their parental propertiesin unique and, often beneficial, ways; however, some may be incapable ofpackaging which, in effect, reduces the diversity of the library.Diversity concentration of the library is achieved through peptideinsertion techniques such as, without limitation, iii-iv) above.Diversity of the library is also concentrated in techniques such as v)above, and such concentration is directed onto multiple hypervariableregions, which lie on surface exposed loops, of the AAV capsid. Whilemany of the techniques generate variant capsids with only a small areaof the capsid mutated, these techniques can be paired with additionalmutagenesis strategies to modify the full capsid.

Once the AAV library or libraries is/are generated, viruses are thenpackaged, such that each AAV particle is comprised of a mutant capsidsurrounding a cap gene encoding that capsid, and purified. Variants ofthe library are then subjected to in vitro and/or in vivo selectivepressure techniques known by and readily available to the skilledartisan in the field of AAV. See e.g., Maheshri, N. et al. NatureBiotech. 24, 198-204 (2006); Dalkara, D. et al. Sci. Transl. Med. 5,189ra76 (2013); Lisowski, L. et al. Nature. 506, 382-286 (2013); Yang,L. et al. PNAS. 106, 3946-3951 (2009); Gao, G. et al. Mol. Ther. 13,77-87 (2006); and Bell, P. et al. Hum. Gene. Ther. 22, 985-997 (2011).For example, without limitation, AAV variants can be selected using i)affinity columns in which elution of different fractions yields variantswith altered binding properties; ii) primary cells—isolated from tissuesamples or immortal cells lines that mimic the behavior of cells in thehuman body—which yield AAV variants with increased efficiency and/ortissue specificity; iii) animal models—which mimic a clinical genetherapy environment—which yield AAV variants that have successfullyinfected target tissue; iv) human xenograft models which yield AAVvariants that have infected grafted human cells; and/or a combination ofselection techniques thereof.

Once viruses are selected, they may be recovered by known techniquessuch as, without limitation, adenovirus-mediated replication, PCRamplification, Next Generation sequencing and cloning, and the like.Virus clones are then enriched through repeated rounds of the selectiontechniques and AAV DNA is isolated to recover selected variant cap genesof interest. Such selected variants can be subjected to furthermodification or mutation and as such serve as a new starting point forfurther selection steps to iteratively increase AAV viral fitness.However, in certain instances, successful capsids have been generatedwithout additional mutation.

The AAV variants disclosed herein were generated at least in partthrough the use of in vivo directed evolution methodology, such as thetechniques described above, involving the use of primate retinal screensfollowing intravitreal administration. As such, he AAV variant capsidsdisclosed herein comprise one or more modifications in amino acidsequence that confer more efficient transduction of primate retinalcells than a corresponding parental AAV capsid protein. As used herein,a “corresponding parental AAV capsid protein” refers to an AAV capsidprotein of the same wild-type or variant AAV serotype as the subjectvariant AAV capsid protein but that does not comprise the one or moreamino acid sequence modifications of the subject variant AAV capsidprotein.

In some embodiments, the subject variant AAV capsid protein comprises aheterologous peptide of from about 5 amino acids to about 20 amino acidsinserted by covalent linkage into an AAV capsid protein GH loop, or loopIV, relative to a corresponding parental AAV capsid protein. By the “GHloop,” or loop IV, of the AAV capsid protein it is meant thesolvent-accessible portion referred to in the art as the GH loop, orloop IV, of AAV capsid protein. For the GH loop/loop IV of AAV capsid,see, e.g., van Vliet et al. (2006) Mol. Ther. 14:809; Padron et al.(2005) J. Virol. 79:5047; and Shen et al. (2007) Mol. Ther. 15:1955.Thus, for example, the insertion site can be within about amino acids411-650 of an AAV VP1 capsid protein. For example, the insertion sitecan be within amino acids 571-612 of AAV1 VP1, amino acids 570-611 ofAAV2 VP1, within amino acids 571-612 of AAV3A VP1, within amino acids571-612 of AAV3B VP1, within amino acids 569-610 of AAV4 VP1, withinamino acids 560-601 of AAV5 VP1, within amino acids 571 to 612 of AAV6VP1, within amino acids 572 to 613 of AAV7 VP1, within amino acids 573to 614 of AAV8 VP1, within amino acids 571 to 612 of AAV9 VP1, or withinamino acids 573 to 614 of AAV10 VP1, or the corresponding amino acids ofany variant thereof. Those skilled in the art would know, based on acomparison of the amino acid sequences of capsid proteins of various AAVserotypes, where an insertion site “corresponding to amino acids ofAAV2” would be in a capsid protein of any given AAV serotype. See alsoFIG. 6 for an alignment of wild-type AAV SEQ ID NOS:1-11 which providesamino acid locations between and across the wild-type (naturallyoccurring) serotypes AAV1, AAV2, AAV3A, AAV3B, and AAV4-10.

In certain embodiments, the insertion site is a single insertion sitebetween two adjacent amino acids located between amino acids 570-614 ofVP1 of any wild-type AAV serotype or AAV variant, e.g., the insertionsite is between two adjacent amino acids located in amino acids 570-610,amino acids 580-600, amino acids 570-575, amino acids 575-580, aminoacids 580-585, amino acids 585-590, amino acids 590-600, or amino acids600-614, of VP1 of any AAV serotype or variant. For example, theinsertion site can be between amino acids 580 and 581, amino acids 581and 582, amino acids 583 and 584, amino acids 584 and 585, amino acids585 and 586, amino acids 586 and 587, amino acids 587 and 588, aminoacids 588 and 589, or amino acids 589 and 590. The insertion site can bebetween amino acids 575 and 576, amino acids 576 and 577, amino acids577 and 578, amino acids 578 and 579, or amino acids 579 and 580. Theinsertion site can be between amino acids 590 and 591, amino acids 591and 592, amino acids 592 and 593, amino acids 593 and 594, amino acids594 and 595, amino acids 595 and 596, amino acids 596 and 597, aminoacids 597 and 598, amino acids 598 and 599, or amino acids 599 and 600.For example, the insertion site can be between amino acids 587 and 588of AAV2, between amino acids 590 and 591 of AAV1, between amino acids588 and 589 of AAV3A, between amino acids 588 and 589 of AAV3B, betweenamino acids 584 and 585 of AAV4, between amino acids 575 and 576 ofAAV5, between amino acids 590 and 591 of AAV6, between amino acids 589and 590 of AAV7, between amino acids 590 and 591 of AAV8, between aminoacids 588 and 589 of AAV9, or between amino acids 588 and 589 of AAV10.

In some embodiments, a peptide insertion disclosed herein has a lengthof 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 aminoacids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids,14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 aminoacids, 19 amino acids, or 20 amino acids. In another embodiment, apeptide insertion disclosed herein comprises from 1 to 4 spacer aminoacids at the amino terminus (N-terminus) and/or at the carboxyl terminus(C-terminus) of any one of the peptide insertions disclosed herein.Exemplary spacer amino acids include, without limitation, leucine (L),alanine (A), glycine (G), serine (S), threonine (T), and proline (P). Incertain embodiments, a peptide insertion comprises 2 spacer amino acidsat the N-terminus and 2 spacer amino acids at the C-terminus. In otherembodiments, a peptide insertion comprises 2 spacer amino acids at theN-terminus and 1 spacer amino acids at the C-terminus.

The peptide insertions disclosed herein have not been previouslydescribed and/or inserted into an AAV capsid. Without wishing to bebound by theory, the presence of any of the disclosed peptide insertionsmay act to lower the variant capsid's affinity for heparin sulfate whichlikely reduces binding to the extracellular matrix in the front of theprimate retina. In addition, the peptide insertion motifs disclosedherein may confer enhanced transduction of primate retinal cells throughthe addition of a cell surface receptor binding domain.

In some preferred embodiments, the insertion peptide comprises an aminoacid sequence of any one of the formulas below.

In some aspects, an insertion peptide can be a peptide of 7 to 10 aminoacids in length, of Formula 1a:

Y₁Y₂X₁X₂X₃X₄X₅X₆X₇Y₃

-   -   Where each of Y₁-Y₃, if present, is independently selected from        Ala, Leu, Gly, Ser, Thr, Pro    -   X₁ is selected from Gln, Asn, His, Ile, and Ala    -   X₂ is selected from Ala, Gln, Asp, Ser, Lys, and Pro    -   X₃ is selected from Asp, Ile, Thr and Asn    -   X₄ is selected from Thr, Ser, Tyr, Gln, Glu, and Ala    -   X₅ is selected from Thr, Lys, and Asn    -   X₆ is selected from Lys, Asn, and Glu    -   X₇ is selected from Asn, Thr, Ile, His, Asp, and Ala.

In certain embodiments, the insertion peptide of Formula 1a comprises anamino acid sequence selected from QADTTKN (SEQ ID NO:13), ISDQTKH (SEQID NO:14), ASDSTKA (SEQ ID NO:15), NQDYTKT (SEQ NO:16), HDITKNI (SEQ IDNO:17), HPDTTKN (SEQ ID NO:18), HQDTTKN (SEQ ID NO:19), NKTTNKD (SEQ IDNO:20), ISNENEH (SEQ ID NO:21), and QANANEN (SEQ ID NO:22).

In other aspects, an insertion peptide can be a peptide of 7 to 10 aminoa acids in length, of Formula 1b:

Y₁Y₂X₁X₂X₃X₄X₅X₆X₇Y₃

-   -   Where each of Y₁-Y₃, if present, is independently selected from        Ala, Leu, Gly, Ser, Thr, Pro    -   X₁ is selected from Gln, Asn, His, and Ile    -   X₂ is selected from Ala, Gin, Asp, and Ser    -   X₃ is selected from Asp and Ile    -   X₄ is selected from Thr, Tyr, and Gln    -   X₅ is selected from Thr and Lys    -   X₆ is selected from Lys and Asn    -   X₇ is selected from Asn, Thr, Ile, and His

In certain embodiments, the insertion peptide of Formula 1b comprises anamino acid sequence selected from QADTTKN (SEQ ID NO:13), ISDQTKH (SEQID NO:14), NQDYTKT (SEQ NO:16), HDITKNI (SEQ ID NO:17), and HQDTTKN (SEQID NO:19).

In other aspects, an insertion peptide can be a peptide of 7 to 10 aminoacids in length, of Formula 1c

Y₁Y₂X₁X₂AspX₃ThrLysX₄Y₃

-   -   Where each of Y₁-Y₃, if present, is independently selected from        Ala, Leu, Gly, Ser, Thr, Pro    -   X₁ is selected from Gln, Asn, His, and Ile    -   X₂ is selected from Ala, Gln, and Ser    -   X₃ is selected from Thr, Tyr, and Gln    -   X₄ is selected from Asn, Thr, and His

In certain embodiments, the insertion peptide of Formula 1c comprises anamino acid sequence selected from QADTTKN (SEQ ID NO:13), ISDQTKH (SEQID NO:14), NQDYTKT (SEQ NO:16), and HQDTTKN (SEQ 1D NO:19).

In other aspects, an insertion peptide can be a peptide of 7 to 10 aminoacids in length, of Formula 1d:

Y₁Y₂X₁X₂AspX₃ThrX₄Y₃

-   -   Where each of Y₁-Y₃, if present, is independently selected from        Ala, Leu, Gly, Ser, Thr, Pro    -   X₁ is selected from Gln and Ile    -   X₂ is selected from Ala and Ser    -   X₃ is selected from Thr and Gln    -   X₄ is selected from Asn and His

In certain embodiments, the insertion peptide of Formula 1d comprises anamino acid sequence selected from QADTTKN (SEQ ID NO:13) and ISDQTKH(SEQ ID NO:14).

In other aspects, an insertion peptide can be a peptide of 7 to 11 aminoacids in length, of Formula Ie

Y₁Y₂X₁X₂AsnX₃AsnGluX₄Y₃

-   -   Where each of Y₁-Y₃, if present, is independently selected from        Ala, Leu, Gly, Ser, Thr, Pro    -   X₁ is selected from Gln and Ile    -   X₂ is selected from Ala and Ser    -   X₃ is selected from Glu and Ala    -   X₄ is selected from Asn and His

In other embodiments, an insertion peptide of Formula 1e comprises anamino acid sequence selected from ISNENEH (SEQ ID NO:21), and QANANEN(SEQ ID NO:22).

In yet another embodiment, an insertion peptide can be a peptide of 7 to11 amino acids in length, of Formula IIa:

Y₁Y₂X₁X₂DX₃TKX₄Y₃.

-   -   Wherein each of Y₁-Y₃, if present, is independently selected        from Ala, Leu, Gly, Ser, Thr, Pro    -   X₁ is selected from Q, N, A, H, and I;    -   X₂ is selected from Q, A, P, and S;    -   X₃ is selected from T, Y, S, and Q; and    -   X₄ is selected from T, N, A, and H.

In a further embodiment of a peptide insertion of an amino acid sequenceof the formula X₁X₂DX₃TKX₄, the peptide insertion is selected from thegroup consisting of QADTTKN (SEQ ID NO:13), ISDQTKH (SEQ ID NO:14),ASDSTKA, NQDYTKT (SEQ NO:16), HQDTTKN (SEQ ID NO:19), and HPDTTKN (SEQID NO:18).

In some such embodiments, an insertion peptide can be a peptide of 7 to11 amino acids in length, of Formula IIb:

Y₁Y₂X₁X₂DX₃TKX₄Y₃

-   -   Wherein each of Y₁-Y₃, if present, is independently selected        from Ala, Leu, Gly, Ser, Thr, Pro    -   X₁ is selected from N, A, and H;    -   X₂ is selected from Q, P, and S;    -   X₃ is selected from T, Y, and S; and    -   X₄ is selected from T, N, and A.

In a further embodiment of a peptide insertion of an amino acid sequenceof the formula X₁X₂DX₃TKX₄, the peptide insertion is selected from thegroup consisting of ASDSTKA, NQDYTKT (SEQ NO:16), HQDTTKN (SEQ IDNO:19), and HPDTTKN (SEQ ID NO:18).

In other embodiments, the insertion peptide comprises an amino acidsequence selected from KDRAPST (SEQ ID NO:26), TNRTSPD (SEQ ID NO:24),PNSTHGS (SEQ ID NO:25) and GKSKVID (SEQ ID NO:23).

In some embodiments, the insertion peptide comprises an amino acidsequence selected from ASDSTKA (SEQ ID NO:15), QANANEN (SEQ ID NO:22),QADTTKN (SEQ ID NO:13), ISDQTKH (SEQ ID NO:14), NQDYTKT (SEQ ID NO:16),HDITKNI (SEQ ID NO:17), HPDTTKN (SEQ ID NO:18), HQDTTKN (SEQ ID NO:19),NKTTNKD (SEQ ID NO:20), ISNENEH (SEQ ID NO:21), GKSKVID (SEQ ID NO:23),TNRTSPD (SEQ ID NO:24), PNSTHGS (SEQ ID NO:25) and KDRAPST (SEQ IDNO:26).

In other preferred embodiments, the insertion peptide has from 1 to 3spacer amino acids (Y₁-Y₃) at the amino and/or carboxyl terminus of anamino acid sequence selected from QADTTKN (SEQ ID NO:13), ISDQTKH (SEQID NO:14), ASDSTKA (SEQ ID NO:15), NQDYTKT (SEQ ID NO:16), HDITKNI (SEQID NO:17), HPDTTKN (SEQ ID NO:18), HQDTTKN (SEQ ID NO:19), NKTTNKD (SEQID NO:20), ISNENEH (SEQ ID NO:21), QANANEN (SEQ ID NO:22), GKSKVID (SEQID NO:23), TNRTSPD (SEQ ID NO:24), PNSTHGS (SEQ ID NO:25) and KDRAPST(SEQ ID NO:26). In certain such embodiments, the insertion peptide isselected from the group consisting of: LAQADTTKNA (SEQ ID NO:27),LAISDQTKHA (SEQ ID NO:28), LGISDQTKHA (SEQ ID NO:29), LAASDSTKAA (SEQ IDNO:30), LANQDYTKTA (SEQ ID NO:31), LAHDITKNIA (SEQ ID NO:32), LAHPDTTKNA(SEQ ID NO:33), LAHQDTKNA (SEQ ID NO:34), LANKTTNKDA (SEQ ID NO:35),LPISNENEHA (SEQ ID NO:36), LPQANANENA (SEQ ID NO:37), LAGKSKVIDA (SEQ IDNO:38), LATNRTSPDA (SEQ ID NO:39), LAPNSTHGSA (SEQ ID NO:40) andLAKDRAPSTA (SEQ ID NO:41).

In some embodiments, the subject variant AAV capsid protein does notinclude any other amino acid sequence modifications other than a peptideinsertion of from about 5 amino acids to about 20 amino acids in the GHloop, or loop IV. For example, in some embodiments, the subject variantAAV capsid protein comprises a peptide insertion comprising an aminoacid sequence selected from the group consisting of QADTTKN (SEQ IDNO:13), ISDQTKH (SEQ ID NO:14), ASDSTKA (SEQ ID NO:15), NQDYTKT (SEQ IDNO:16), HDITKNI (SEQ ID NO:17), HPDTTKN (SEQ ID NO:18) HQDTTKN (SEQ IDNO:19), NKTTNKD (SEQ ID NO:20), ISNENEH (SEQ ID NO:21), QANANEN (SEQ IDNO:22), GKSKVID (SEQ ID NO:23), TNRTSPD (SEQ ID NO:24), PNSTHGS (SEQ IDNO:25), KDRAPST (SEQ ID NO:26), LAQADTTKNA (SEQ ID NO:27), LAISDQTKHA(SEQ ID NO:28), LGISDQTKHA (SEQ ID NO:29), LAASDSTKAA (SEQ ID NO:30),LANQDYTKTA (SEQ ID NO:31), LAHDITKNIA (SEQ ID NO:32), LAHPDTTKNA (SEQ IDNO:33), LAHQDTTKNA (SEQ ID NO:34), LANKTTNKDA (SEQ ID NO:35), LPISNENEHA(SEQ ID NO:36), LPQANANENA (SEQ ID NO:37), LAGKSKVIDA (SEQ ID NO:38),LATNRTSPDA (SEQ ID NO:39), LAPNSTHGSA (SEQ ID NO:40) and LAKDRAPSTA (SEQID NO:41), and the variant AAV capsid does not include any other aminoacid substitutions, insertions, or deletions (i.e., the variant AAVcapsid protein comprises said insertion and is otherwise identical tothe corresponding AAV capsid protein). Put another way, the variant AAVcapsid protein comprising said insertion is otherwise identical to theparental AAV capsid protein into which the peptide has been inserted. Asanother example, the subject variant AAV capsid protein comprises apeptide insertion having an amino acid sequence selected from QADTTKN(SEQ ID NO:13), ISDQTKH (SEQ ID NO:14), ASDSTKA (SEQ ID NO:15), NQDYTKT(SEQ ID NO:16), HDITKNI (SEQ ID NO:17), HPDTTKN (SEQ ID NO:18), HQDTTKN(SEQ ID NO:19), NKTTNKD (SEQ ID NO:20), ISNENEH (SEQ ID NO:21), QANANEN(SEQ ID NO:22), GKSKVID (SEQ ID NO:23), TNRTSPD (SEQ ID NO:24), PNSTHGS(SEQ ID NO:25), KDRAPST (SEQ ID NO:26), LAQADTTKNA (SEQ ID NO:27),LAISDQTKHA (SEQ ID NO:28), LGISDQTKHA (SEQ ID NO:29), LAASDSTKAA (SEQ IDNO:30), LANQDYTKTA (SEQ ID NO:31), LAHDITKNIA (SEQ ID NO:32), LAHPDTTKNA(SEQ ID NO:33), LAHQDTTKNA (SEQ ID NO:34), LANKTTNKDA (SEQ ID NO:35),LPISNENEHA (SEQ ID NO:36), LPQANANENA (SEQ ID NO:37), LAGKSKVIDA (SEQ IDNO:38), LATNRTSPDA (SEQ ID NO:39), LAPNSTHGSA (SEQ ID NO:40) andLAKDRAPSTA (SEQ ID NO:41), wherein the peptide insertion is locatedbetween amino acids 587 and 588 of the VP1 of the AAV2 capsid or thecorresponding amino acids of a VP1 of another parental AAV, e.g. betweenamino acids 588 and 589 of VP1 of AAV1, AAV3A, AAV3B, AAV6 or AAV9,between amino acids 586 and 587 of VP1 of AAV4, between amino acids 577and 578 of VP1 of AAV5, between amino acids 589 and 590 of VP1 of AAV7,between amino acids 590 to 591 of VP1 of AAV8 or AAV10, etc. wherein thevariant AAV capsid protein sequence is otherwise identical to thecorresponding parental AAV capsid protein sequence, e.g. any one of SEQID NOs:1-12.

In other embodiments, the subject variant AAV capsid protein, inaddition to comprising a peptide insertion, e.g. as disclosed herein oras known in the art, in the GH loop, comprises from about 1 to about 100amino acid substitutions or deletions, e.g. 1 to about 5, from about 2to about 4, from about 2 to about 5, from about 5 to about 10, fromabout 10 to about 15, from about 15 to about 20, from about 20 to about25, from about 25-50, from about 50-100 amino acid substitutions ordeletions compared to the parental AAV capsid protein. Thus, in someembodiments, a subject variant capsid protein comprises an amino acidsequence having a sequence identity of 85% or more, 90% or more, 95% ormore, or 98% or more, e.g. or 99% identity to the corresponding parentalAAV capsid, e.g. a wild type capsid protein as set forth in SEQ IDNOs:1-12.

In a further embodiment, the one or more amino acid substitutions are atamino acid residue(s) 1, 15, 34, 57, 66, 81, 101, 109, 144, 164, 176,188, 196, 226, 236, 240, 250, 312, 363, 368, 449, 456, 463, 472, 484,524, 535, 551, 593, 698, 708, 719, 721, and/or 735 of AAV2 VP1 capsidprotein as numbered prior to insertion of the peptide, or thecorresponding amino acid residue(s) of another AAV capsid protein. Insome such embodiments, the one or more amino acid substitutions areselected from the group consisting of M1L, L15P, P34A, N57D, N66K, R81Q,Q101R, S109T, R144K, R144M, Q164K, T176P, L188I, S196Y, G226E, G236V,I240T, P250S, N312K, P363L, D368H, N449D, T456K, S463Y, D472N, R484C,A524T, P535S, N551S, A593E, I698V, V708I, V719M, S721L, and L735Q ofAAV2 VP1 capsid protein as numbered prior to the insertion of thepeptide, or the corresponding amino acid residue(s) of another AAVcapsid protein.

In a preferred embodiment, a variant AAV capsid protein is providedcomprising a) a peptide insertion in the GH-loop of the capsid protein,wherein the peptide insertion comprises an amino acid sequence selectedfrom ISDQTKH (SEQ ID NO:14), LGISDQTKHA (SEQ ID NO:29) and LAISDQTKHA(SEQ ID NO:28), and b) one or more of the following amino acidsubstitutions compared to the amino acid sequence of AAV2 (SEQ ID NO:2)or the corresponding substitution in another AAV parental serotype (i.e.other than AAV2), wherein the substituted amino acid(s) do not naturallyoccur at the corresponding positions: M1L, L15P, P34A, N57D, N66K, R81Q,Q101R, S109T, R144K, R144M, Q164K, T176P, 188I, S196Y, G226E, G236V,I240T, P250S, N312K, P363L, D368H, N449D, T456K, S463Y, D472N, R484C,A524T, P535S, N551S, A593E, I698V, V708I, V719M, S721L, L735Q and acombination thereof. In some embodiments, the one or more amino acidsubstitutions are selected from the group consisting of: M1L+L15P+P535S,P34A, P34A+S721L, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K,Q164K+V708I, T176P, L188I, S196Y, G226E, G236V, I240T, N312K,N312K+N449D+D472N+N551S+I698V+L735Q, P363L, R484C+V708I, T456K andV708I. Preferably, the peptide insertion site is located between aminoacids 587 and 588 of AAV2 capsid or the corresponding position in thecapsid protein of another AAV serotype.

In a particularly preferred embodiment, the variant AAV capsid comprisesa peptide insertion comprising the amino acid sequence ISDQTKH (SEQ IDNO:14) or comprising, consisting essentially of, or consisting of theamino acid sequence LAISDQTKHA (SEQ ID NO:28) or LGISDQTKHA (SEQ IDNO:29) between amino acids 587 and 588 of VP1 of AAV2 or thecorresponding amino acids of another AAV capsid, and further comprises aP34A amino acid substitution at residue 34 relative to the amino acidsequence of AAV2 capsid (SEQ ID NO:2) or the corresponding residue ofanother AAV capsid. The variant AAV capsid may have at least about 85%,at least about 90%, at least about 95%, at least about 98%, or at leastabout 99%, or greater, amino acid sequence identity to the entire lengthof the amino acid sequence set forth in SEQ ID NO:2 or the correspondingparental AAV capsid. In a particularly preferred embodiment, the variantAAV capsid has an amino acid sequence having at least about 85%, atleast about 90%, at least about 95%, at least about 98% sequenceidentity to or is 100% identical to the following amino acid sequence:

(SEQ ID NO: 42) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPK A AERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYNTSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LAISDQTKHA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

In another particularly preferred embodiment, the variant AAV capsidcomprises a peptide insertion comprising the amino acid sequence ISDQTKH(SEQ ID NO:14) or comprising, consisting essentially of, or consistingof the amino acid sequence LAISDQTKHA (SEQ ID NO:28) or LGISDQTKHA (SEQID NO:29) between amino acids 587 and 588 of AAV2 capsid protein or thecorresponding position in the capsid protein of another AAV serotype andcomprises an N312K amino acid substitution compared to the amino acidsequence of AAV2 capsid (SEQ ID NO:2) or the corresponding substitutionin another AAV parental serotype and optionally further comprises N449D,D472N, N551S, I698V and/or L735Q amino acid substitutions compared tothe amino acid sequence of AAV2 capsid or the correspondingsubstitutions in another AAV parental serotype. In another particularlypreferred embodiment, the variant AAV capsid comprises a peptideinsertion comprising the amino acid sequence ISDQTKH (SEQ ID NO:14) orcomprising, consisting essentially of, or consisting of the amino acidsequence LAISDQTKHA (SEQ ID NO:28) or LGISDQTKHA (SEQ ID NO:29) betweenamino acids 587 and 588 of AAV2 capsid or the corresponding position inthe capsid protein of another AAV serotype and comprises N312K, N449D,D472N, N551S, I698V and L735Q amino acid substitutions compared to theamino acid sequence of AAV2 capsid (SEQ ID NO:2) or substitutions at thecorresponding residues in another AAV parental serotype. The variant AAVcapsid may have at least about 85%, at least about 90%, at least about95%, at least about 98%, or greater, amino acid sequence identity to theentire length of the amino acid sequence set forth in SEQ ID NO:2. In aparticularly preferred embodiment, the variant AAV capsid has an aminoacid sequence having at least about 85%, at least about 90%, at leastabout 95%, at least about 98% sequence identity to or is 100% identicalto the following amino acid sequence:

(SEQ ID NO: 43) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLI NNNWGFRP KRLKFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFRADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRT D TPSGTTTQSRLQFSQAGASDIR N QSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKT SVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LAISDQTKHA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKEN SKRWNPE VQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRN Q

In another embodiment, a variant AAV capsid protein is providedcomprising a) a peptide insertion located between amino acids 588 and589 of VP1 of AAV1, AAV3A, AAV3B, AAV6 or AAV9, amino acids 586 and 587of AAV4, amino acids 577 and 578 of AAV5, amino acids 589 and 590 ofAAV7, or amino acids 590 to 591 of AAV8 or AAV10, the peptide insertioncomprising an amino acid sequence selected from ISDQTKH (SEQ ID NO:14),LGISDQTKHA (SEQ ID NO:29) and LAISDQTKHA (SEQ ID NO:28), and b) a valineto isoleucine substitution at amino acid 709 of AAV3A or AAV3B, analanine to isoleucine substitution at position 709 of AAV1 or AAV6, anasparagine to isoleucine substitution at amino acid 707 of AAV4 or aminoacid 709 of AAV9 or a threonine to isoleucine substitution at amino acid710 of AAV7 or amino acid 711 of AAV8 or AAV10 or a glutamine toisoleucine substitution at amino acid 697 of AAV5 and is optionallyotherwise identical to any one of SEQ ID NOs: 1 and 3-12. In preferredembodiments, the variant capsid protein comprises a) a peptide insertioncomprising the amino acid sequence ISDQTKH (SEQ ID NO:14) or comprising,consisting essentially of, or consisting of the amino acid sequenceLAISDQTKHA (SEQ ID NO:28) or LGISDQTKHA (SEQ ID NO:29) between aminoacids 587 and 588 of AAV2 capsid and b) a valine to isoleucine aminoacid substitution at amino acid 708 compared to the amino acid sequenceof AAV2, wherein the variant capsid protein comprises from 2 to 5, from5 to 10, or from 10 to 15 amino acid substitutions.

In yet another embodiment, the variant capsid protein comprises a) apeptide insertion comprising the amino acid sequence ISDQTKH (SEQ IDNO:14) or comprising, consisting essentially of, or consisting of theamino acid sequence LAISDQTKHA (SEQ ID NO:28) or LGISDQTKHA (SEQ IDNO:29) between amino acids 587 and 588 of AAV2 capsid and b) a valine toisoleucine amino acid substitution at amino acid 708 compared to theamino acid sequence of AAV2 and is otherwise identical to the amino acidsequence of SEQ ID NO:2.

In yet another embodiment, the variant capsid protein comprises a) apeptide insertion comprising the amino acid sequence ISDQTKH (SEQ IDNO:14) or comprising, consisting essentially of, or consisting of theamino acid sequence LAISDQTKHA (SEQ ID NO:28) or LGISDQTKHA (SEQ IDNO:29) between amino acids 587 and 588 of AAV2 capsid and is otherwiseidentical to the amino acid sequence of SEQ ID NO:2. In someembodiments, the variant AAV capsid has an amino acid sequence having atleast about 85%, at least about 90%, at least about 95%, at least about98% sequence identity to or is 100% identical to the following aminoacid sequence:

(SEQ ID NO: 44) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTFWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTINNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LGISDQTKHA RQAATADVNTQGVLGPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

In a preferred embodiment, a variant AAV capsid protein is providedcomprising a) a peptide insertion in the GH-loop of the capsid protein,wherein the peptide insertion comprises an amino acid sequence selectedfrom QADTTKN (SEQ ID NO:13) and LAQADTTKNA (SEQ ID NO:27), and b) one ormore of the following amino acid substitutions compared to the aminoacid sequence of AAV2 (SEQ ID NO:2) or the corresponding substitution inanother AAV parental serotype (i.e. other than AAV2), wherein thesubstituted amino acid(s) do not naturally occur at the correspondingpositions: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K,R144M, Q164K, T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K,P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S,A593E, I698V, V719M, S721L, L735Q and a combination thereof, preferablyselected from S109T, P250S, A524T, A593E, I698V, V708I, and/or V719M.The peptide insertion site is preferably located between amino acids 587and 588 of AAV2 capsid or the corresponding position in the capsidprotein of another AAV serotype. In a particularly preferred embodiment,the variant AAV capsid comprises a peptide insertion comprising theamino acid sequence QADTTKN (SEQ ID NO:13) or comprising, consistingessentially of, or consisting of the amino acid sequence LAQADTTKNA (SEQID NO:27) between amino acids 587 and 588 of AAV2 capsid or thecorresponding position in the capsid protein of another AAV serotype andcomprises an I698V amino acid substitution compared to the amino acidsequence of AAV2 or the corresponding substitution in another AAVparental serotype, wherein the substituted amino acid(s) do notnaturally occur at the corresponding position. The variant AAV capsidmay have at least about 85%, at least about 90%, at least about 95%, atleast about 98%, or greater, amino acid sequence identity to the entirelength of the amino acid sequence set forth in SEQ ID NO:2. In someembodiments, the corresponding amino acid substitution is an I699V aminoacid substitution compared to the amino acid sequence of AAV3A, AAV3B orAAV9 capsid, an 1687V substitution compared to the amino acid sequenceof AAV5 capsid, an I700V substitution compared to the amino acidsequence of AAV7, an I701V substitution compared to the amino acidsequence of AAV8 or AAV10. In a particularly preferred embodiment, thevariant AAV capsid has an amino acid sequence having at least about 85%,at least about 90%, at least about 95%, at least about 98% sequenceidentity to or is 100% identical to the following amino acid sequence:

(SEQ ID NO: 45) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LAQADTTKNA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKEN SKRWNPE VQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

In other preferred embodiments, the variant AAV capsid comprises apeptide insertion comprising the amino acid sequence QADTTKN (SEQ IDNO:13) or comprising, consisting essentially of, or consisting of theamino acid sequence LAQADTTKNA (SEQ ID NO:27) between amino acids 587and 588 of AAV2 capsid or the corresponding position in the capsidprotein of another AAV serotype and comprises a V719M amino acidsubstitution and optionally a V708I substitution compared to the aminoacid sequence of AAV2 or the corresponding substitution in another AAVparental serotype, wherein the substituted amino acid(s) do notnaturally occur at the corresponding position.

In another embodiment, a variant AAV capsid protein is providedcomprising a) a peptide insertion located between amino acids 588 and589 of VP1 of AAV1, AAV3A, AAV3B, AAV6 or AAV9, amino acids 586 and 587of AAV4, amino acids 577 and 578 of AAV5, amino acids 589 and 590 ofAAV7, or amino acids 590 to 591 of AAV8 or AAV10, the peptide insertioncomprising an amino acid sequence selected from QADTTKN (SEQ ID NO:13)and LAQADTTKNA (SEQ ID NO:27), and b) a valine to isoleucinesubstitution at amino acid 709 of AAV3A or AAV3B, an alanine toisoleucine substitution at position 709 of AAV1 or AAV6, an asparagineto isoleucine substitution at amino acid 707 of AAV4 or amino acid 709of AAV9 or a threonine to isoleucine substitution at amino acid 710 ofAAV7 or amino acid 711 of AAV8 or AAV10 or a glutamine to isoleucinesubstitution at amino acid 697 of AAV5. In another embodiment, a variantAAV capsid protein is provided comprising a) a peptide insertion locatedbetween amino acids 588 and 589 of VP1 of AAV1, AAV3A, AAV3B, AAV6 orAAV9, amino acids 586 and 587 of AAV4, amino acids 577 and 578 of AAV5,amino acids 589 and 590 of AAV7, or amino acids 590 to 591 of AAV8 orAAV10, the peptide insertion comprising an amino acid sequence selectedfrom QADTTKN (SEQ ID NO:13) and LAQADTITKNA (SEQ ID NO:27), and b) aserine to threonine amino acid substitution at position 109 compared tothe amino acid sequence of AAV1, AAV3A, AAV3B, AAV4, AAV7, AAV8, AAV9,or AAV10 or at position 108 compared to the amino acid sequence of AAV5or AAV6. In preferred embodiments, the variant AAV capsid comprises apeptide insertion comprising the amino acid sequence QADTTKN (SEQ IDNO:13) or comprising, consisting essentially of, or consisting of theamino acid sequence LAQADTTKNA (SEQ ID NO:27) between amino acids 587and 588 of AAV2 capsid and comprises a serine to threonine substitutionat amino acid 109 (S109T) or a valine to isoleucine amino acidsubstitution at amino acid 708 (V708I) compared to the amino acidsequence of AAV2, wherein the variant capsid protein comprises from 1 to5, from 5 to 10, or from 10 to 15 amino acid substitutions and ispreferably at least about 85%, at least about 90%, at least about 95%,at least about 98%, or greater amino acid sequence identity to theentire length of the amino acid sequence set forth in SEQ ID NO:2. Inother preferred embodiments, the variant AAV capsid comprises a peptideinsertion comprising the amino acid sequence QADTTKN (SEQ ID NO:13) orcomprising, consisting essentially of, or consisting of the amino acidsequence LAQADTTKNA (SEQ ID NO:27) between amino acids 587 and 588 ofAAV2 capsid or the corresponding position in the capsid protein ofanother AAV serotype and comprises a serine to threonine substitution atamino acid 109 and a valine to isoleucine amino acid substitution atamino acid 708 compared to the amino acid sequence of AAV2.

In yet another embodiment, the variant capsid protein comprises a) apeptide insertion comprising the amino acid sequence QADTTKN (SEQ IDNO:13) or comprising, consisting essentially of, or consisting of theamino acid sequence LAQADTTKNA (SEQ ID NO:27) between amino acids 587and 588 of AAV2 capsid and b) at least one amino acid substitution,wherein the amino acid sequence of the variant capsid does not comprisea valine to isoleucine amino acid substitution at amino acid 708compared to the amino acid sequence of AAV2 and does not comprise aserine to threonine substitution at amino acid 109 compared to the aminoacid sequence of AAV2.

In yet another embodiment, the variant capsid protein comprises a) apeptide insertion comprising the amino acid sequence QADTTKN (SEQ IDNO:13) or comprising, consisting essentially of, or consisting of theamino acid sequence LAQADTTKNA (SEQ ID NO:27) between amino acids 587and 588 of AAV2 capsid and is otherwise identical to the amino acidsequence of SEQ ID NO:2.

In another preferred embodiment, a variant AAV capsid protein isprovided comprising a) a peptide insertion in the GH-loop of the capsidprotein, wherein the peptide insertion comprises an amino acid sequenceselected from HDITKNI (SEQ ID NO:17), IAHDITKNIA (SEQ ID NO:60) andLAHDITKNIA (SEQ ID NO:32), and b) one or more of the following aminoacid substitutions compared to the amino acid sequence of AAV2 (SEQ IDNO:2) or the corresponding substitution in another AAV parental serotype(i.e. other than AAV2), wherein the substituted amino acid(s) do notnaturally occur at the corresponding positions: M1L, L15P, P34A, N57D,N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K, T176P, L188I, S196Y,G226E, G236V, I240T, P250S, N312K, P363L, D368H, R389S, N449D, T456K,S463Y, D472N, R484C, A524T, P535S, N551S, A593E, I698V, V708I, V719M,S721L, L735Q and a combination thereof. In some embodiments, the AAVcapsid protein comprises one or more amino acid substitutions selectedfrom S109T, R389S, A593E and/or V708I. Preferably, the peptide insertionsite is located between amino acids 587 and 588 of AAV2 capsid or thecorresponding position in the capsid protein of another AAV serotype. Inone preferred embodiment, the variant AAV capsid comprises a peptideinsertion comprising the amino acid sequence HDITKNI (SEQ ID NO:17) orcomprising, consisting essentially of, or consisting of the amino acidsequence IAHDITKNIA (SEQ ID NO:60) or LAHDITKNIA (SEQ ID NO:32) betweenamino acids 587 and 588 of AAV2 capsid and comprises an S109T amino acidsubstitution compared to the amino acid sequence of AAV2 capsid or thecorresponding substitution in another AAV parental serotype. The variantAAV capsid may have at least about 85%, at least about 90%, at leastabout 95%, at least about 98%, or greater amino acid sequence identityto the entire length of the amino acid sequence set forth in SEQ IDNO:2.

In yet another embodiment, the variant capsid comprises a) a peptideinsertion comprising the amino acid sequence HDITKNI (SEQ ID NO:17) orcomprising, consisting essentially of, or consisting of the amino acidsequence IAHDITKNIA (SEQ ID NO:60) or LAHDITKNIA (SEQ ID NO:32) betweenamino acids 587 and 588 of AAV2 capsid and is otherwise identical to theamino acid sequence set forth in SEQ ID NO:2. In some embodiments, thevariant AAV capsid has an amino acid sequence having at least about 85%,at least about 90%, at least about 95%, at least about 98% sequenceidentity to or is 100% identical to the following amino acid sequence:

(SEQ ID NO: 46) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPRAAPSGLGTNTMATGSGAPNIADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LAHDITKNIA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

In other embodiments, the variant capsid comprises a) a peptideinsertion comprising, consisting essentially of, or consisting of theamino acid sequence LAHDITKNIA between amino acids 587 and 588 of AAV2capsid and b) at least one amino acid substitution, wherein the aminoacid sequence of the variant capsid does not comprise a valine toisoleucine amino acid substitution at amino acid 708 compared to theamino acid sequence of AAV2. In yet other embodiments, the variantcapsid comprises a) a peptide insertion comprising the amino acidsequence DITKNIA (SEQ ID NO:61) or comprising, consisting essentially ofor consisting of the amino acid sequence IAHDITKNIA (SEQ ID NO:60) orLAHDITKNIA (SEQ ID NO:32) between amino acids 587 and 588 of AAV2 capsidand b) a V708I substitution compared to the amino acid sequence of AAV2.In other embodiments, the variant capsid comprises a) a peptideinsertion comprising, consisting essentially of, or consisting of theamino acid sequence LAHDITKNIA (SEQ ID NO:32) between amino acids 587and 588 of AAV2 capsid and b) two or more amino acid substitutions,wherein the amino acid sequence of the variant capsid comprises a valineto isoleucine amino acid substitution at amino acid 708 compared to theamino acid sequence of AAV2.

In another preferred embodiment, a variant AAV capsid protein isprovided comprising a) a peptide insertion in the GH-loop of the capsidprotein, wherein the peptide insertion comprises an amino acid sequenceselected from NQDYTKT (SEQ ID NO:16) and LANQDYTKTA (SEQ ID NO:31), andb) one or more of the following amino acid substitutions compared to theamino acid sequence of AAV2 (SEQ ID NO:2) or the correspondingsubstitution in another AAV parental serotype (i.e. other than AAV2),wherein the substituted amino acid(s) do not naturally occur at thecorresponding positions: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R,S109T, R144K, R144M, Q164K, T176P, L188I, S196Y, G226E, G236V, I240T,P250S, P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S,N551S, A593E, I698V, V708I, V719M, S721L, L735Q and a combinationthereof. In some embodiments, the AAV capsid protein comprises one ormore amino acid substitutions selected from S109T, S109T+S463Y, D368Hand V708I. Preferably, the peptide insertion site is located betweenamino acids 587 and 588 of AAV2 capsid or the corresponding position inthe capsid protein of another AAV serotype. In one preferred embodiment,the variant AAV capsid comprises a peptide insertion comprising theamino acid sequence NQDYTKT (SEQ ID NO:16) or comprising, consistingessentially of, or consisting of the amino acid sequence LANQDYTKTA (SEQID NO:31) between amino acids 587 and 588 of AAV2 capsid and comprises aV708I amino acid substitution compared to the amino acid sequence ofAAV2 capsid or the corresponding substitution in another AAV parentalserotype. The variant AAV capsid may have at least about 85%, at leastabout 90%, at least about 95%, at least about 98%, or greater amino acidsequence identity to the entire length of the amino acid sequence setforth in SEQ ID NO:2. In yet another embodiment, the variant capsidcomprises a) a peptide insertion comprising the amino acid sequenceNQDYTKT (SEQ ID NO:16) or comprising, consisting essentially of, orconsisting of the amino acid sequence LANQDYTKTA (SEQ ID NO:31) betweenamino acids 587 and 588 of AAV2 capsid and is otherwise identical to theamino acid sequence set forth in SEQ ID NO:2. In some embodiments, thevariant AAV capsid has an amino acid sequence having at least about 85%,at least about 90%, at least about 95%, at least about 98% sequenceidentity to or is 100% identical to the following amino acid sequence:

(SEQ ID NO: 47) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LANQDYTKTA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSIGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNI

In other embodiments, the variant capsid comprises a) a peptideinsertion comprising the amino acid sequence NQDYTKT (SEQ ID NO:16) orcomprising, consisting essentially of, or consisting of the amino acidsequence LANQDYTKTA (SEQ ID NO:31) between amino acids 587 and 588 ofAAV2 capsid and b) an S109T amino acid substitution compared to thesequence of SEQ ID NO:2 and optionally an S463Y amino acid substitution,wherein the variant capsid is at least about 85%, at least about 90%, atleast about 95%, at least about 98% identical to the entire length ofthe amino acid sequence set forth in SEQ ID NO:2. In relatedembodiments, the variant capsid comprises a) a peptide insertioncomprising the amino acid sequence NQDYTKT (SEQ ID NO:16) or comprising,consisting essentially of, or consisting of the amino acid sequenceLANQDYTKTA (SEQ ID NO:31) between amino acids 587 and 588 of AAV2 capsidand b) an S109T amino acid substitution compared to the amino acidsequence of SEQ ID NO:2 and is otherwise identical to the amino acidsequence of SEQ ID NO:2.

In another embodiment, a variant AAV capsid protein is providedcomprising a) a peptide insertion located between amino acids 588 and589 of VP1 of AAV1, AAV3A, AAV3B, AAV6 or AAV9, amino acids 586 and 587of AAV4, amino acids 577 and 578 of AAV5, amino acids 589 and 590 ofAAV7, or amino acids 590 to 591 of AAV8 or AAV10, the peptide insertioncomprising an amino acid sequence selected from NQDYTKT (SEQ ID NO:16)and LANQDYTKTA (SEQ ID NO:31), and b) an asparagine to lysine amino acidsubstitution at position 313 compared to the amino acid sequence of AAV1or AAV6, or at position 314 compared to the amino acid sequence of AAV9,or a serine to lysine substitution at position 312 of AAV3A or AAV3B orat position 315 of AAV8 or AAV10, or an arginine to lysine substitutionat position 303 of AAV4 or AAV5, or at position 314 of AAV7. In anotherembodiment, the variant capsid comprises a) a peptide insertioncomprising the amino acid sequence NQDYTKT (SEQ ID NO:16) or comprising,consisting essentially of or consisting of the amino acid sequenceLANQDYTKTA (SEQ ID NO:31) between amino acids 587 and 588 of AAV2 capsidand b) an N312K amino acid substitution, wherein the variant capsidprotein comprises from 1 to 5, from 5 to 10, or from 10 to 15 amino acidsubstitutions.

In another embodiment, a variant AAV capsid protein is providedcomprising a) a peptide insertion in the GH-loop of the capsid protein,wherein the peptide insertion comprises an amino acid sequence selectedfrom PNSTHGS (SEQ ID NO:25) and LAPNSTHGSA (SEQ ID NO:40), and b) one ormore of the following amino acid substitutions compared to the aminoacid sequence of AAV2 (SEQ ID NO:2) or the corresponding substitution inanother AAV parental serotype (i.e. other than AAV2), wherein thesubstituted amino acid(s) do not naturally occur at the correspondingpositions: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K,R144M, Q164K, T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K,P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S,A593E, I698V, V708I, V719M, S721L, L735Q and a combination thereof.Preferably, the peptide insertion site is located between amino acids587 and 588 of AAV2 capsid or the corresponding position in the capsidprotein of another AAV serotype. In one preferred embodiment, thevariant AAV capsid comprises a peptide insertion comprising the aminoacid sequence PNSTHGS (SEQ ID NO:25) or comprising, consistingessentially of, or consisting of the amino acid sequence LAPNSTHGSA (SEQID NO:40) between amino acids 587 and 588 of AAV2 capsid and comprises aV708I amino acid substitution compared to the amino acid sequence ofAAV2 capsid or the corresponding substitution in another AAV parentalserotype. The variant AAV capsid may have at least about 85%, at leastabout 90%, at least about 95%, at least about 98%, or greater amino acidsequence identity to the entire length of the amino acid sequence setforth in SEQ ID NO:2. In yet another embodiment, the variant capsidcomprises a) a peptide insertion comprising the amino acid sequencePNSTHGS (SEQ ID NO:25) or comprising, consisting essentially of, orconsisting of the amino acid sequence LAPNSTHGSA (SEQ ID NO:40) betweenamino acids 587 and 588 of AAV2 capsid and is otherwise identical to theamino acid sequence set forth in SEQ ID NO:2. In some embodiments, thevariant AAV capsid has an amino acid sequence having at least about 85%,at least about 90%, at least about 95%, at least about 98% sequenceidentity to or is 100% identical to the following amino acid sequence:

(SEQ ID NO: 48) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDCTTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LAPNSTHGSA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

In another embodiment, a variant AAV capsid protein is providedcomprising a) a peptide insertion in the GH-loop of the capsid protein,wherein the peptide insertion comprises an amino acid sequence selectedfrom NKTTNKDA (SEQ ID NO:62) and LANKTTNKDA (SEQ ID NO:35), and b) oneor more of the following amino acid substitutions compared to the aminoacid sequence of AAV2 (SEQ ID NO:2) or the corresponding substitution inanother AAV parental serotype (i.e. other than AAV2), wherein thesubstituted amino acid(s) do not naturally occur at the correspondingpositions: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K,R144M, Q164K, T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K,P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S,A593E, I698V, V708I, V719M, S721L, L735Q and a combination thereof.Preferably, the peptide insertion site is located between amino acids587 and 588 of AAV2 capsid or the corresponding position in the capsidprotein of another AAV serotype. In one preferred embodiment, thevariant AAV capsid comprises a peptide insertion comprising the aminoacid sequence NKTTNKDA (SEQ ID NO:62) or comprising, consistingessentially of, or consisting of the amino acid sequence LANKTTNKDA (SEQID NO:35) between amino acids 587 and 588 of AAV2 capsid and comprisesan N449D amino acid substitution compared to the amino acid sequence ofAAV2 capsid or the corresponding substitution in another AAV parentalserotype. The variant AAV capsid may have at least about 85%, at leastabout 90%, at least about 95%, at least about 98%, or greater amino acidsequence identity to the entire length of the amino acid sequence setforth in SEQ ID NO:2. In yet another embodiment, the variant capsidcomprises a) a peptide insertion comprising the amino acid sequenceNKTTNKDA (SEQ ID NO:62) or comprising, consisting essentially of, orconsisting of the amino acid sequence LANKTTNKDA (SEQ ID NO:35) betweenamino acids 587 and 588 of AAV2 capsid and is otherwise identical to theamino acid sequence set forth in SEQ ID NO:2. In some embodiments, thevariant AAV capsid has an amino acid sequence having at least about 85%,at least about 90%, at least about 95%, at least about 98% sequenceidentity to or is 100% identical to the following amino acid sequence:

(SEQ ID NO: 49) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDPNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMTNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LANKTTNKDA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

In another embodiment, a variant AAV capsid protein is providedcomprising a) a peptide insertion in the GH-loop of the capsid protein,wherein the peptide insertion comprises an amino acid sequence selectedfrom TNRTSPD (SEQ ID NO:24) and LATNRTSPDA (SEQ ID NO:39), and b) one ormore of the following amino acid substitutions compared to the aminoacid sequence of AAV2 (SEQ ID NO:2) or the corresponding substitution inanother AAV parental serotype (i.e. other than AAV2), wherein thesubstituted amino acid(s) do not naturally occur at the correspondingpositions: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K,R144M, Q164K, T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K,P363L, D368H N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S,A593E, I698V, V719M, S721L, L735Q and a combination thereof. Preferably,the peptide insertion site is located between amino acids 587 and 588 ofAAV2 capsid or the corresponding position in the capsid protein ofanother AAV serotype. In a related embodiment, a variant AAV capsidprotein is provided comprising a) a peptide insertion located betweenamino acids 588 and 589 of VP1 of AAV1, AAV3A, AAV3B, AAV6 or AAV9,amino acids 586 and 587 of AAV4, amino acids 577 and 578 of AAV5, aminoacids 589 and 590 of AAV7, or amino acids 590 to 591 of AAV8 or AAV10,the peptide insertion comprising an amino acid sequence selected fromTNRTSPD (SEQ ID NO:24) and LATNRTSPDA (SEQ ID NO:39), and b) a valine toisoleucine substitution at amino acid 709 of AAV3A or AAV3B, an alanineto isoleucine substitution at position 709 of AAV1 or AAV6, anasparagine to isoleucine substitution at amino acid 707 of AAV4 or aminoacid 709 of AAV9 or a threonine to isoleucine substitution at amino acid710 of AAV7 or amino acid 711 of AAV8 or AAV10 or a glutamine toisoleucine substitution at amino acid 697 of AAV5. In other embodiments,the variant capsid protein comprises a) a peptide insertion comprising,consisting essentially of, or consisting of the amino acid sequenceLATNRTSPDA (SEQ ID NO:39) between amino acids 587 and 588 of AAV2 capsidand b) a valine to isoleucine amino acid substitution at amino acid 708compared to the amino acid sequence of AAV2, wherein the variant capsidprotein comprises from 1 to 5, from 5 to 10, or from 10 to 15 amino acidsubstitutions. In yet another embodiment, the variant capsid proteincomprises a) a peptide insertion comprising the amino acid sequenceTNRTSPD (SEQ ID NO:24) between amino acids 587 and 588 of AAV2 capsidand b) a valine to isoleucine amino acid substitution at amino acid 708compared to the amino acid sequence of AAV2. The variant AAV capsid mayhave at least about 85%, at least about 90%, at least about 95%, atleast about 98%, or greater amino acid sequence identity to the entirelength of the amino acid sequence set forth in SEQ ID NO:2.

In yet another embodiment, the variant capsid protein comprises a) apeptide insertion comprising the amino acid sequence TNRTSPD (SEQ IDNO:24) or comprising, consisting essentially of, or consisting of theamino acid sequence LATNRTSPDA (SEQ ID NO:39) between amino acids 587and 588 of AAV2 capsid and is otherwise identical to the amino acidsequence of SEQ ID NO:2. In some embodiments, the variant AAV capsid hasan amino acid sequence having at least about 85%, at least about 90%, atleast about 95%, at least about 98% sequence identity to or is 100%identical to the following amino acid sequence:

(SEQ ID NO: 50) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPQYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLCQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFRADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LATNRTSPDA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

In another embodiment, a variant AAV capsid protein is providedcomprising a) a peptide insertion in the GH-loop of the capsid protein,wherein the peptide insertion comprises an amino acid sequence selectedfrom GKSKVID (SEQ ID NO:23) and LAGKSKVIDA (SEQ ID NO:38), and b) one ormore of the following amino acid substitutions compared to the aminoacid sequence of AAV2 (SEQ ID NO:2) or the corresponding substitution inanother AAV parental serotype (i.e. other than AAV2), wherein thesubstituted amino acid(s) do not naturally occur at the correspondingpositions: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K,R144M, Q164K, T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K,P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S,A593E, I698V, V708I, V719M, S721L, L735Q and a combination thereof.Preferably, the peptide insertion site is located between amino acids587 and 588 of AAV2 capsid or the corresponding position in the capsidprotein of another AAV serotype. The variant AAV capsid may have atleast about 85%, at least about 90%, at least about 95%, at least about98%, or greater amino acid sequence identity to the entire length of theamino acid sequence set forth in SEQ ID NO:2. In some embodiments, thevariant AAV capsid comprises a peptide insertion located between aminoacids 587 and 588 of AAV2 capsid comprising the amino acid sequenceGKSKVID (SEQ ID NO:23) or comprising, consisting essentially of orconsisting of the amino acid sequence LAGKSKVIDA (SEQ ID NO:38) and isotherwise identical to the amino acid sequence of SEQ ID NO:2. In otherembodiments, the variant AAV capsid comprises a) a peptide insertioncomprising, consisting essentially of, or consisting of the amino acidsequence LAGKSKVIDA (SEQ ID NO:38) between amino acids 587 and 588 ofAAV2 capsid and comprises at least one amino acid substitution.

In some embodiments, the variant AAV capsid has an amino acid sequencehaving at least about 85%, at least about 90%, at least about 95%, atleast about 98% sequence identity to or is 100% identical to thefollowing amino acid sequence:

(SEQ ID NO: 51) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKRAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LAGKSKVIDA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

In another embodiment, a variant AAV capsid protein is providedcomprising a) a peptide insertion in the GH-loop of the capsid protein,wherein the peptide insertion comprises an amino acid sequence selectedfrom ASDSTKA (SEQ ID NO:15) and LAASDSTKAA (SEQ ID NO:30), and b) one ormore of the following amino acid substitutions compared to the aminoacid sequence of AAV2 (SEQ ID NO:2) or the corresponding substitution inanother AAV parental serotype (i.e. other than AAV2), wherein thesubstituted amino acid(s) do not naturally occur at the correspondingpositions: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K,R144M, Q164K, T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K,P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S,A593E, I698V, V708I, V719M, S721L, L735Q and a combination thereof.Preferably, the peptide insertion site is located between amino acids587 and 588 of AAV2 capsid or the corresponding position in the capsidprotein of another AAV serotype. The variant AAV capsid may have atleast about 85%, at least about 90%, at least about 95%, at least about98%, or greater amino acid sequence identity to the entire length of theamino acid sequence set forth in SEQ ID NO:2. In yet another embodiment,the variant capsid comprises a peptide insertion comprising the aminoacid sequence ASDSTKA (SEQ ID NO:15) or comprising, consistingessentially of, or consisting of the amino acid sequence LAASDSTKAA (SEQID NO:30) between amino acids 587 and 588 of AAV2 capsid and isotherwise identical to the amino acid sequence set forth in SEQ ID NO:2.In some embodiments, the variant AAV capsid has an amino acid sequencehaving at least about 85%, at least about 90%, at least about 95%, atleast about 98% sequence identity to or is 100% identical to thefollowing amino acid sequence:

(SEQ ID NO: 52) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LAASDSTKAA RQAATADVNTQGVLPQMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

In another embodiment, a variant AAV capsid protein is providedcomprising a) a peptide insertion in the GH-loop of the capsid protein,wherein the peptide insertion comprises an amino acid sequence selectedfrom KDRAPST (SEQ ID NO:26) and LAKDRAPTSA (SEQ ID NO:41), and b) one ormore of the following amino acid substitutions compared to the aminoacid sequence of AAV2 (SEQ ID NO:2) or the corresponding substitution inanother AAV parental serotype (i.e. other than AAV2), wherein thesubstituted amino acid(s) do not naturally occur at the correspondingpositions: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K,R144M, Q164K, T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K,P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S,A593E, I698V, V708I, V719M, S721L, L735Q and a combination thereof.Preferably, the peptide insertion site is located between amino acids587 and 588 of AAV2 capsid or the corresponding position in the capsidprotein of another AAV serotype. The variant AAV capsid may have atleast about 85%, at least about 90%, at least about 95%, at least about98%, or greater amino acid sequence identity to the entire length of theamino acid sequence set forth in SEQ ID NO:2. In yet another embodiment,the variant capsid comprises a peptide insertion comprising the aminoacid sequence KDRAPST (SEQ ID NO:26) or comprising, consistingessentially of, or consisting of the amino acid sequence LAKDRAPTSA (SEQID NO:41) between amino acids 587 and 588 of AAV2 capsid and isotherwise identical to the amino acid sequence set forth in SEQ ID NO:2.In some embodiments, the variant AAV capsid has an amino acid sequencehaving at least about 85%, at least about 90%, at least about 95%, atleast about 98% sequence identity to or is 100% identical to thefollowing amino acid sequence:

(SEQ ID NO: 53) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LAKDRAPSTA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

In another embodiment, a variant AAV capsid protein is providedcomprising a) a peptide insertion in the GH-loop of the capsid protein,wherein the peptide insertion comprises an amino acid sequence selectedfrom HQDTTKN (SEQ ID NO:19) and LAHQDTTKNA (SEQ ID NO:34), and b) one ormore of the following amino acid substitutions compared to the aminoacid sequence of AAV2 (SEQ ID NO:2) or the corresponding substitution inanother AAV parental serotype (i.e. other than AAV2), wherein thesubstituted amino acid(s) do not naturally occur at one or more of thecorresponding positions: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R,S109T, R144K, R144M, Q164K, T176P, L188I, S196Y, G226E, G236V, I240T,P250S, N312K, P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T,P535S, N551S, A593E, I698V, V708I, V719M, S721L, L735Q and a combinationthereof. Preferably, the peptide insertion site is located between aminoacids 587 and 588 of AAV2 capsid or the corresponding position in thecapsid protein of another AAV serotype. The variant AAV capsid may haveat least about 85%, at least about 90%, at least about 95%, at leastabout 98%, or greater amino acid sequence identity to the entire lengthof the amino acid sequence set forth in SEQ ID NO:2. In yet anotherembodiment, the variant capsid comprises a peptide insertion comprisingthe amino acid sequence HQDTTKN (SEQ ID NO:19) or comprising, consistingessentially of, or consisting of the amino acid sequence LAHQDTTKNA (SEQID NO:34) between amino acids 587 and 588 of AAV2 capsid and isotherwise identical to the amino acid sequence set forth in SEQ ID NO:2.In some embodiments, the variant AAV capsid has an amino acid sequencehaving at least about 85%, at least about 90%, at least about 95%, atleast about 98% sequence identity to or is 100% identical to thefollowing amino acid sequence:

(SEQ ID NO: 54) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LAHQDTTKNA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

In another embodiment, a variant AAV capsid protein is providedcomprising a) a peptide insertion in the GH-loop of the capsid protein,wherein the peptide insertion comprises an amino acid sequence selectedfrom ISNENEH (SEQ ID NO:21) and LPISNENEHA (SEQ ID NO:36), and b) one ormore of the following amino acid substitutions compared to the aminoacid sequence of AAV2 (SEQ ID NO:2) or the corresponding substitution inanother AAV parental serotype (i.e. other than AAV2), wherein thesubstituted amino acid(s) do not naturally occur at one or more of thecorresponding positions: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R,S109T, R144K, R144M, Q164K, T176P, L188I, S196Y, G226E, G236V, I240T,P250S, N312K, P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T,P535S, N551S, A593E, I698V, V708I, V719M, S721L, L735Q and a combinationthereof. Preferably, the peptide insertion site is located between aminoacids 587 and 588 of AAV2 capsid or the corresponding position in thecapsid protein of another AAV serotype. The variant AAV capsid may haveat least about 85%, at least about 90%, at least about 95%, at leastabout 98%, or greater amino acid sequence identity to the entire lengthof the amino acid sequence set forth in SEQ ID NO:2. In yet anotherembodiment, the variant capsid comprises a peptide insertion comprisingthe amino acid sequence ISNENEH (SEQ ID NO:21) or comprising, consistingessentially of, or consisting of the amino acid sequence LPISNENEHA (SEQID NO:36) between amino acids 587 and 588 of AAV2 capsid and isotherwise identical to the amino acid sequence set forth in SEQ ID NO:2.In some embodiments, the variant AAV capsid has an amino acid sequencehaving at least about 85%, at least about 90%, at least about 95%, atleast about 98% sequence identity to or is 100% identical to thefollowing amino acid sequence:

(SEQ ID NO: 55) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNYKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFRSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LPISNENEHA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

In another embodiment, a variant AAV capsid protein is providedcomprising a) a peptide insertion in the GH-loop of the capsid protein,wherein the peptide insertion comprises an amino acid sequence selectedfrom QANANEN (SEQ ID NO:22) and LPQANANENA (SEQ ID NO:37), and b) one ormore of the following amino acid substitutions compared to the aminoacid sequence of AAV2 (SEQ ID NO:2) or the corresponding substitution inanother AAV parental serotype (i.e. other than AAV2), wherein thesubstituted amino acid(s) do not naturally occur at the correspondingpositions: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K,R144M, Q164K, T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K,P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S,A593E, I698V, V708I, V719M, S721L, L735Q and a combination thereof.Preferably, the peptide insertion site is located between amino acids587 and 588 of AAV2 capsid or the corresponding position in the capsidprotein of another AAV serotype. The variant AAV capsid may have atleast about 85%, at least about 90%, at least about 95%, at least about98%, or greater amino acid sequence identity to the entire length of theamino acid sequence set forth in SEQ ID NO:2. In yet another embodiment,the variant capsid comprises a peptide insertion comprising the aminoacid sequence QANANEN (SEQ ID NO:22) or comprising, consistingessentially of, or consisting of the amino acid sequence LPQANANENA (SEQID NO:37) between amino acids 587 and 588 of AAV2 capsid and isotherwise identical to the amino acid sequence set forth in SEQ ID NO:2.In some embodiments, the variant AAV capsid has an amino acid sequencehaving at least about 85%, at least about 90%, at least about 95%, atleast about 98% sequence identity to or is 100% identical to thefollowing amino acid sequence:

(SEQ ID NO: 56) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LPQANANENA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNI

In another embodiment, a variant AAV capsid protein is providedcomprising a) a peptide insertion in the GH-loop of the capsid protein,wherein the peptide insertion comprises an amino acid sequence selectedfrom HPDTTKN (SEQ ID NO:18) and LAHPDTTKNA (SEQ ID NO:33), and b) one ormore of the following amino acid substitutions compared to the aminoacid sequence of AAV2 (SEQ ID NO:2) or the corresponding substitution inanother AAV parental serotype (i.e. other than AAV2), wherein thesubstituted amino acid(s) do not naturally occur at the correspondingpositions: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K,R144M, Q164K, T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K,P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S,A593E, I698V, V708I, V719M, S721L, L735Q and a combination thereof.Preferably, the peptide insertion site is located between amino acids587 and 588 of AAV2 capsid or the corresponding position in the capsidprotein of another AAV serotype. The variant AAV capsid may have atleast about 85%, at least about 90%, at least about 95%, at least about98%, or greater amino acid sequence identity to the entire length of theamino acid sequence set forth in SEQ ID NO:2. In yet another embodiment,the variant capsid comprises a peptide insertion comprising the aminoacid sequence HPDTTKN (SEQ ID NO:18) or comprising, consistingessentially of, or consisting of the amino acid sequence LAHPDTTKNA (SEQID NO:33) between amino acids 587 and 588 of AAV2 capsid and isotherwise identical to the amino acid sequence set forth in SEQ ID NO:2.In some embodiments, the variant AAV capsid has an amino acid sequencehaving at least about 85%, at least about 90%, at least about 95%, atleast about 98% sequence identity to or is 100% identical to thefollowing amino acid sequence:

(SEQ ID NO: 57) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LAHPDTTKNA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL.

In several aspects, a variant AAV capsid protein is provided comprisingone or more amino acid substitutions relative to a correspondingparental AAV capsid protein, wherein the variant capsid protein, whenpresent in an AAV virion, confers increased infectivity of a retinalcell compared to the infectivity of a retinal cell by an AAV virioncomprising the corresponding parental AAV capsid protein.

In some embodiments, a variant AAV capsid protein comprises a P34A aminoacid substitution compared to the amino acid sequence of AAV2 capsid(SEQ ID NO:2) or a P33A amino acid substitution compared to the aminoacid sequence of AAV5 capsid (SEQ ID NO:6). In some preferredembodiments, the variant capsid protein comprises an amino acid sequencehaving at least about 85%, at least about 90%, at least about 95%, atleast about 98%, or at least about 99%, or greater, amino acid sequenceidentity to the entire length of the amino acid sequence set forth inSEQ ID NO:2 or SEQ ID NO:6 and comprises a P34A or P33A amino acidsubstitution compared to the amino acid sequence of AAV2 or AAV5 capsidrespectively. In some preferred embodiments, the variant capsid proteincomprises an amino acid sequence comprising a P34A amino acidsubstitution compared to the amino acid sequence set forth in SEQ ID NO2 and is otherwise identical to the amino acid sequence set forth in SEQID NO:2. In related embodiments, the variant capsid protein comprises aP34A amino acid substitution compared to the amino acid sequence of SEQID NO:2, wherein the variant capsid protein comprises from 1 to 5, from5 to 10, or from 10 to 15 amino acid substitutions compared to the aminoacid sequence of an AAV2 capsid protein set forth in SEQ ID NO:2.

In other embodiments a variant AAV capsid protein comprises an aminoacid substitution at amino acid 164 compared to the amino acid sequenceof AAV2 capsid (SEQ ID NO:2) or the corresponding position in anotherAAV parental serotype (i.e. other than AAV2), wherein the substitutedamino acid does not naturally occur at the corresponding position. Insome preferred embodiments, the variant capsid protein comprises anamino acid sequence having at least about 85%, at least about 90%, atleast about 95%, at least about 98%, or at least about 99%, or greater,amino acid sequence identity to the entire length of the amino acidsequence set forth in SEQ ID NO 2 and comprises an amino acidsubstitution at amino acid 164 compared to the amino acid sequence ofAAV2 capsid (SEQ ID NO:2). In some embodiments, the rAAV virioncomprises a glutamine to lysine amino acid substitution at amino acid164 compared to amino acid sequence of AAV1, AAV2 or AAV6 or at aminoacid 165 compared to the amino acid sequence of AAV7, AAV8, or AAV10; orcomprises a serine to lysine substitution at amino acid 160 of AAV5 orcomprises an alanine to lysine substitution at amino acid 164 of AAV9.In related embodiments, the variant capsid protein comprises an aminoacid substitution at amino acid 164 (e.g. Q164K) compared to the aminoacid sequence of AAV2 capsid (SEQ ID NO:2), wherein the variant capsidprotein comprises from 1 to 5, from 5 to 10, or from 10 to 15 amino acidsubstitutions compared to the amino acid sequence of an AAV2 capsidprotein set forth in SEQ ID NO:2. In some preferred embodiments, thevariant capsid protein comprises an amino acid sequence comprising aQ164K amino acid substitution compared to the amino acid sequence setforth in SEQ ID NO:2 and is otherwise identical to the amino acidsequence set forth in SEQ ID NO:2. In other embodiments the variantcapsid protein comprises Q164K and V708I amino acid substitutionscompared to the amino acid sequence of AAV2 capsid (SEQ ID NO:2) or thecorresponding substitutions in another AAV parental serotype (i.e. otherthan AAV2) and is at least about 85%, at least about 90%, at least about95%, at least about 98%, or at least about 99%, or greater, amino acidsequence identity to the entire length of the amino acid sequence setforth in SEQ ID NO:2.

In other embodiments a variant AAV capsid protein comprises an aminoacid substitution at amino acid 698 compared to the amino acid sequenceof AAV2 capsid (SEQ ID NO:2) or the corresponding position in anotherAAV parental serotype (i.e. other than AAV2), wherein the substitutedamino acid does not naturally occur at the corresponding position. Insome preferred embodiments, the variant capsid protein comprises anamino acid sequence having at least about 85%, at least about 90%, atleast about 95%, at least about 98%, or at least about 99%, or greater,amino acid sequence identity to the entire length of the amino acidsequence set forth in SEQ ID NO 2 and comprises an amino acidsubstitution at amino acid 698 compared to the amino acid sequence ofAAV2 capsid (SEQ ID NO:2). In some embodiments, the rAAV virioncomprises an isoleucine to valine amino acid substitution at amino acid698 compared to amino acid sequence of AAV2, or at amino acid 699compared to the amino acid sequence of AAV3A, AAV3B, or AAV9, or atamino acid 687 of AAV5, or at amino acid 700 of AAV7, or at amino acid701 of AAV8 or AAV10. In related embodiments, the variant capsid proteincomprises an amino acid substitution at amino acid 699 (e.g. I698V)compared to the amino acid sequence of AAV2 capsid (SEQ ID NO:2),wherein the variant capsid protein comprises from 1 to 5, from 5 to 10,or from 10 to 15 amino acid substitutions compared to the amino acidsequence of an AAV2 capsid protein set forth in SEQ ID NO:2. In somepreferred embodiments, the variant capsid protein comprises an aminoacid sequence comprising an I698V amino acid substitution compared tothe amino acid sequence set forth in SEQ ID NO:2 and is otherwiseidentical to the amino acid sequence set forth in SEQ ID NO:2.

In other embodiments a variant AAV capsid protein comprises an aminoacid substitution at amino acid 109 compared to the amino acid sequenceof AAV2 capsid (SEQ ID NO:2) or the corresponding position in anotherAAV parental serotype (i.e. other than AAV2). In some preferredembodiments, the variant capsid protein comprises an amino acid sequencehaving at least about 85%, at least about 90, at least about 95%, atleast about 98%, or at least about 99%, or greater, amino acid sequenceidentity to the entire length of the amino acid sequence set forth inSEQ ID NO 2 and comprises an amino acid substitution at amino acid 109compared to the amino acid sequence of AAV2 capsid (SEQ ID NO:2). Insome embodiments, the variant capsid protein comprises a serine tothreonine amino acid substitution at position 109 compared to the aminoacid sequence of AAV1, AAV3A, AAV3B, AAV4, AAV7, AAV8, AAV9, or AAV10 orat position 108 compared to the amino acid sequence of AAV5 or AAV6. Inrelated embodiments, the variant capsid protein comprises an S109T aminoacid substitution compared to the amino acid sequence AAV2, wherein thevariant capsid protein comprises from 1 to 5, from 5 to 10, or from 10to 15 amino acid substitutions. In other related embodiments, thevariant capsid protein comprises an S109T amino acid substitution and anA593E amino acid substitution compared to the amino acid sequence ofAAV2. In some embodiments the variant capsid protein comprises S109T andA493V and optionally A593E and/or V708I amino acid substitutionscompared to the amino acid sequence of AAV2 capsid (SEQ ID NO:2) or thecorresponding substitutions in another AAV parental serotype (i.e. otherthan AAV2) and has at least about 85%, at least about 90%, at leastabout 95%, at least about 98%, or at least about 99%, or greater, aminoacid sequence identity to the entire length of the amino acid sequenceset forth in SEQ ID NO 2. In some preferred embodiments the variantcapsid protein comprises S109T, A493V, A593E and V708I amino acidsubstitutions compared to the amino acid sequence of AAV2 capsid (SEQ IDNO:2) or the corresponding substitutions in another AAV parentalserotype (i.e. other than AAV2) and is at least about 85%, at leastabout 90%, at least about 95%, at least about 98%, or at least about99%, or greater, amino acid sequence identity to the entire length ofthe amino acid sequence set forth in SEQ ID NO 2. In other preferredembodiments, the variant capsid protein comprises S109T and V708I aminoacid substitutions compared to the amino acid sequence of AAV2 capsidand has at least about 85%, at least about 90%, at least about 95%, atleast about 98%, or at least about 99%, or greater, amino acid sequenceidentity to the entire length of the amino acid sequence set forth inSEQ ID NO 2 or is otherwise identical to the amino acid sequence of SEQID NO:2.

In other embodiments a variant AAV capsid protein comprises an aminoacid substitution at amino acid 593 compared to the amino acid sequenceof AAV2 capsid (SEQ ID NO:2) or the corresponding position in anotherAAV parental serotype (i.e. other than AAV2). In some preferredembodiments, the variant capsid protein comprises an amino acid sequencehaving at least about 85%, at least about 90%, at least about 95%, atleast about 99%, or at least about 99%, or greater, amino acid sequenceidentity to the entire length of the amino acid sequence set forth inSEQ ID NO 2 and comprises an amino acid substitution at amino acid 593compared to the amino acid sequence of AAV2 capsid (SEQ ID NO:2). Insome embodiments, the variant capsid protein comprises a glycine toglutamate amino acid substitution at amino acid 594 compared to theamino acid sequence of AAV1, AAV3A, AAV6, or AAV9, or at amino acid 583of AAV5, or at amino acid 596 of AAV8 or AAV10, or an arginine toglutamate amino acid substitution at amino acid 594 of AAV3B, or anaspartate to glutamate amino acid substitution at amino acid 592 of AAV4or a glutamine to glutamate amino acid substitution at position 595 ofAAV7. In other embodiments, the variant capsid protein comprises anA593E amino acid substitution compared to the amino acid sequence ofAAV2 and does not comprise one or more of the following amino acidsubstitutions compared to the amino acid sequence of AAV2: I19V, V369A,K26R, N215D, G355S, V46A and S196P. In related embodiments, the variantcapsid protein comprises A593E and N596D amino acid substitutionscompared to the amino acid sequence of AAV2 and has at least about 85%,at least about 90%, at least about 95%, at least about 98% or at leastabout 99% identity to the entire length of the amino acid sequence setforth in SEQ ID NO 2. In other embodiments, the variant capsid comprisesA593E and N596D amino acid substitutions compared to the amino acidsequence of AAV2 and is otherwise identical to the amino acid sequenceof AAV2. In other embodiments, the variant capsid comprises A593E andV708I amino acid substitutions compared to the amino acid sequence ofAAV2 and has at least about 85%, at least about 90%, at least about 95%,at least about 98% or at least about 99% identity to the entire lengthof the amino acid sequence set forth in SEQ ID NO 2. In otherembodiments, the variant capsid comprises A593E and V708I amino acidsubstitutions compared to the amino acid sequence of AAV2 and isotherwise identical to the amino acid sequence of AAV2.

In other embodiments a variant AAV capsid protein comprises an aminoacid substitution at amino acid 708 compared to the amino acid sequenceof AAV2 capsid (SEQ ID NO:2) or the corresponding position in anotherAAV parental serotype (i.e. other than AAV2) wherein the substitutedamino acid does not naturally occur at the corresponding position.Preferably, the rAAV virion does not comprise a proline to serinesubstitution at amino acid 250 compared to AAV2 or a corresponding aminoacid in another AAV parental serotype. In some embodiments, the variantcapsid protein comprises an amino acid sequence having at least about85%, at least about 90%, at least about 95%, at least about 98%, or atleast about 99%, or greater, amino acid sequence identity to the entirelength of the amino acid sequence set forth in SEQ ID NO 2 and comprisesan amino acid substitution at amino acid 708 compared to the amino acidsequence of AAV2 capsid (SEQ ID NO:2). In preferred embodiments, thevariant capsid protein comprises a valine to isoleucine (V708I)substitution at amino acid 708 compared to the amino acid sequence ofAAV2 capsid and has at least about 85%, at least about 90%, at leastabout 95%, at least about 98%, or at least about 99%, or greater, aminoacid sequence identity to the entire length of the amino acid sequenceset forth in SEQ ID NO 2 or is otherwise identical to the amino acidsequence of SEQ ID NO:2, wherein the variant capsid protein does notcomprise a P250S amino acid substitution. In some embodiments, thevariant capsid protein comprises a valine to isoleucine substitution atamino acid 709 of AAV3A or AAV3B, an alanine to isoleucine substitutionat position 709 of AAV1 or AAV6, an asparagine to isoleucinesubstitution at amino acid 707 of AAV4 or amino acid 709 of AAV9 or athreonine to isoleucine substitution at amino acid 710 of AAV7 or aminoacid 711 of AAV8 or AAV10 or a glutamine to isoleucine substitution atamino acid 697 of AAV5. In related embodiments, the variant capsidprotein comprises a V708I amino acid substitution compared to the aminoacid sequence of AAV2, wherein the variant capsid protein comprises from2 to 5, from 5 to 10, or from 10 to 15 amino acid substitutions andwherein the variant capsid protein does not comprise a P250S amino acidsubstitution. In other embodiments, the variant capsid protein comprisesa V708I amino acid substitution and also comprises an A593E and/or anS109T amino acid substitution compared to the amino acid sequence ofAAV2. In other related embodiments, the variant capsid comprises V708Iand A593E amino acid substitutions compared to the amino acid sequenceof AAV2, wherein the variant capsid protein is otherwise identical tothe amino acid sequence of AAV2. In other related embodiments, thevariant capsid comprises V708I and S109T amino acid substitutionscompared to the amino acid sequence of AAV2, wherein the variant capsidprotein is otherwise identical to the amino acid sequence of AAV2. Inother embodiments, the variant capsid protein comprises V708I and V719Mamino acid substitutions compared to the amino acid sequence of AAV2 andhas at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, or at least about 99%, or greater, amino acid sequenceidentity to the entire length of the amino acid sequence set forth inSEQ ID NO 2 or is otherwise identical to the amino acid sequence of SEQID NO:2. In other embodiments, the variant capsid protein comprisesV708I and R733C amino acid substitutions compared to the amino acidsequence of AAV2 and has at least about 85%, at least about 90%, atleast about 95%, at least about 98%, or at least about 99%, or greater,amino acid sequence identity to the entire length of the amino acidsequence set forth in SEQ ID NO 2 or is otherwise identical to the aminoacid sequence of SEQ ID NO:2. In other embodiments, the variant capsidprotein comprises V708I and G727D amino acid substitutions compared tothe amino acid sequence of AAV2 and has at least about 85%, at leastabout 90%, at least about 95%, at least about 98%, or at least about99%, or greater, amino acid sequence identity to the entire length ofthe amino acid sequence set forth in SEQ ID NO 2 or is otherwiseidentical to the amino acid sequence of SEQ ID NO:2.

In other embodiments a variant AAV capsid protein comprises an aminoacid substitution at amino acid 196 compared to the amino acid sequenceof AAV2 capsid (SEQ ID NO:2) or the corresponding position in anotherAAV parental serotype (i.e. other than AAV2), wherein the substitutedamino acid does not naturally occur at the corresponding position and isoptionally other than proline. In some preferred embodiments, thevariant capsid protein comprises an amino acid sequence having at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,or at least about 99%, or greater, amino acid sequence identity to theentire length of the amino acid sequence set forth in SEQ ID NO 2 andcomprises an amino acid substitution at amino acid 196 compared to theamino acid sequence of AAV2 capsid (SEQ ID NO:2) and is optionally otherthan an S196P substitution. In preferred embodiments, the variant capsidprotein comprises a serine to tyrosine amino acid substitution at aminoacid 196 of AAV2 or AAV9 or at amino acid 197 of AAV7, AAV8 or AAV10 orat amino acid 186 of AAV5; or an alanine to tyrosine substitution atamino acid 196 of AAV1 or AAV6; or a methionine to tyrosine substitutionat amino acid 191 of AAV4; or a threonine to tyrosine substitution atamino acid 196 of AAV3A or AAV3B. In a related embodiment, the variantcapsid protein comprises an amino acid sequence comprising an S196Yamino acid substitution compared to the amino acid sequence set forth inSEQ ID NO:2 and is otherwise identical to the amino acid sequence setforth in SEQ ID NO:2. In related embodiments, the variant capsid proteincomprises an amino acid substitution at amino acid 196 other than anS196P substitution (e.g. comprises an S196Y substitution) compared tothe amino acid sequence of AAV2 capsid (SEQ ID NO:2), wherein thevariant capsid protein comprises from 1 to 5, from 5 to 10, or from 10to 15 amino acid substitutions compared to the amino acid sequence of anAAV2 capsid protein set forth in SEQ ID NO:2.

In other embodiments a variant AAV capsid protein comprises an aminoacid substitution at amino acid 175 compared to the amino acid sequenceof AAV2 capsid (SEQ ID NO:2) or the corresponding position in anotherAAV parental serotype (i.e. other than AAV2), wherein the substitutedamino acid does not naturally occur at the corresponding position. Insome preferred embodiments, the variant capsid protein comprises anamino acid sequence having at least about 85%, at least about 90%, atleast about 95%, at least about 98%, or at least about 99%, or greater,amino acid sequence identity to the entire length of the amino acidsequence set forth in SEQ ID NO 2 and comprises an amino acidsubstitution at amino acid 175 compared to the amino acid sequence ofAAV2 capsid (SEQ ID NO:2). In some embodiments, the variant capsidcomprises a Q175H amino acid substitution compared to the amino acidsequence of AAV2 as set forth in SEQ ID NO:2 or a glutamine to histidinesubstitution at the corresponding position in another AAV parentalserotype. In related embodiments, the variant capsid protein comprisesan amino acid substitution at amino acid 175 (e.g. Q175H) compared tothe amino acid sequence of AAV2 capsid (SEQ ID NO:2), wherein thevariant capsid protein comprises from 1 to 5, from 5 to 10, or from 10to 15 amino acid substitutions compared to the amino acid sequence of anAAV2 capsid protein set forth in SEQ ID NO:2.

In other embodiments a variant AAV capsid protein comprises an aminoacid substitution at amino acid 64 compared to the amino acid sequenceof AAV2 capsid (SEQ ID NO:2) or the corresponding position in anotherAAV parental serotype (i.e. other than AAV2), wherein the substitutedamino acid does not naturally occur at the corresponding position. Insome preferred embodiments, the variant capsid protein comprises anamino acid sequence having at least about 85%, at least about 90%, atleast about 95%, at least about 98%, or at least about 99%, or greater,amino acid sequence identity to the entire length of the amino acidsequence set forth in SEQ ID NO 2 and comprises an amino acidsubstitution at amino acid 64 compared to the amino acid sequence ofAAV2 capsid (SEQ ID NO:2). In some embodiments, the rAAV virioncomprises a P64S amino acid substitution compared to the amino acidsequence of AAV2 as set forth in SEQ ID NO:2 or a proline to serinesubstitution at the corresponding position in another AAV parentalserotype. In related embodiments, the variant capsid protein comprisesan amino acid substitution at amino acid 64 (e.g. P64S) compared to theamino acid sequence of AAV2 capsid (SEQ ID NO:2), wherein the variantcapsid protein comprises from 1 to 5, from 5 to 10, or from 10 to 15amino acid substitutions compared to the amino acid sequence of an AAV2capsid protein set forth in SEQ ID NO:2.

In other embodiments, a variant AAV capsid protein comprises an aminoacid sequence at least 85%, at least 90%, at least 95% or at least 98%identical to a wild-type AAV capsid sequence selected from the groupconsisting of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 10, 11 and 12 and alsocomprises i) one or more amino acid substitutions selected from thegroup consisting of P34A, S109T+V708I, A593E+N596D, V708I+V719M,V708I+G727D, S109T+A493V+A593E+V708I, V708I+R733C, Q164K, and I698Vand/or (ii) a peptide insertion selected from the group consisting ofQADTTKN (SEQ ID NO:13), ISDQTKH (SEQ ID NO:14), ASDSTKA (SEQ ID NO:15),NQDYTKT (SEQ ID NO:16), HDITKNI (SEQ ID NO:17), PQANANEN (SEQ ID NO:63),TNRTSPD (SEQ ID NO:24), PNSTHGS (SEQ ID NO:25), KDRAPST (SEQ ID NO:26),HQDTTKN (SEQ ID NO:19), HPDTTKN (SEQ ID NO:18), NKTTNKD (SEQ ID NO:20),GKSKVID (SEQ ID NO:23), PISNENEH (SEQ ID NO:64), LAQADTTKNA (SEQ IDNO:27), LAISDQTKHA (SEQ ID NO:28), LGISDQTKHA (SEQ ID NO:29), LAASDSTKAA(SEQ ID NO:30), LAHDITKNIA (SEQ ID NO:32), LPQANANENA (SEQ ID NO:37),LANQDYTKTA (SEQ ID NO:31), LATNRTSPDA (SEQ ID NO:39), LAPNSTHGSA (SEQ IDNO:40), LAKDRAPSTA (SEQ ID NO:41), LAHQDTTKNA (SEQ ID NO:34), LAHPDTTKNA(SEQ ID NO:33), LANKTTNKDA (SEQ ID NO:35), LAGKSKVIDA (SEQ ID NO:38),and LPISNENEHA (SEQ ID NO:36). In some embodiments, the variant AAVcapsid comprises the specified one or more amino acid substitutionsand/or peptide insertions and is otherwise identical to a sequenceselected from the group consisting of SEQ ID NOS: 1-12.

In some embodiments, a variant AAV capsid protein is an ancestral capsidprotein. By an ancestral capsid protein it is meant an evolutionaryancestor of a capsid protein that is found in nature today, e.g. AAV1,AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAV11, AAV12,AAV13, which is generated in silico by random amino acid substitution atpositions of degeneracy between AAV capsid proteins that are found innature today. One nonlimiting example of an ancestral capsid is providedbelow, wherein the positions of degeneracy (residues 264, 266, 268, 448,459, 460, 467, 470, 471, 474, 495, 516, 533, 547, 551, 555, 557, 561,563, 577, 583, 593, 596, 661, 662, 664, 665, 710, 717, 718, 719, 723)are marked as an “X”:

(SEQ ID NO: 58) MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVERSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPLGEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSXSXGXTNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLXRTQSTGGTAGXXELLFSQXGPXXMSXQAKNWLPGPCYRQQRVSKTLXQNNNSNFAWTGATKYHLNGRXSLVNPGVAMATHKDDEXRFFPSSGVLIFGKXGAGXNNTXLXNVMXTXEEEIKTTNPVATEXYGVVAXNLQSSNTAPXTGXVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPANPPXXFXXAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYAKSXNVDFAVXXXGVYXEPRPIGTRYLTRNL

In some embodiments, the ancestral capsid protein comprises an aminoacid sequence having at least about 85%, at least about 90%, at leastabout 95%, at least about 98%, or at least about 99%, or greater, aminoacid sequence identity to the entire length of the amino acid sequenceset forth in SEQ ID NO:58. In some embodiments, the ancestral capsidprotein comprises an amino acid sequence having at least about 85%, atleast about 90%, at least about 95%, at least about 98%, or at leastabout 99%, or greater, amino acid sequence identity to the entire lengthof the amino acid sequence of AAV2, e.g. as set forth in SEQ ID NO 2. Insome embodiments, the ancestral capsid protein comprises an amino acidsequence having at least about 85%, at least about 90%, at least about95%, at least about 98%, or at least about 99%, or greater, amino acidsequence identity to the entire length of the amino acid sequence of theancestral sequence disclosed in SEQ ID NO:58 or in SEQ ID NO:2 andcomprises one or more amino acid residues selected from the groupconsisting of: Alanine (A) at 264, Alanine (A) at 266, Serine (S) at268, Alanine (A) at 448, Threonine (T) at 459, Arginine (R) at 460,Alanine (A) at 467, Serine (S) at 470, Asparagine (N) at 471, Alanine(A) at 474, Serine (S) at 495, Asparagine (D) at 516, Asparagine (D) at533, Glutamine (Q) at 547, Alanine (A) at 551, Alaninet (A) at 555,Glutamic acid (E) at 557, Methionine (M) at 561, Serine (S) at 563,Glutamine (Q) at 577, Serine (S) at 583, Valine (V) at 593, Threonine T)at 596, Alanine (A) at 661, Valine (V) at 662, Threonine (T) at 664,Proline (P) at 665, Threonine (T) at 710, Aspartic Acid (D) at 717,Asparagine (N) at 718, Glutamic acid (E) at 719, and Serine (S) at 723.In some preferred embodiments, the variant capsid protein comprises anamino acid sequence having at least about 85%, at least about 90%, atleast about 95%, at least about 98%, or at least about 99%, in someinstances 100% amino acid sequence identity to the entire length of thefollowing amino acid sequence and comprises one or more amino acidresidues selected from the group consisting of: Alanine (A) at 264,Alanine (A) at 266, Serine (S) at 268, Alanine (A) at 448, Threonine (T)at 459, Arginine (R) at 460, Alanine (A) at 467, Serine (S) at 470,Asparagine (N) at 471, Alanine (A) at 474, Serine (S) at 495, Asparagine(D) at 516, Asparagine (D) at 533, Glutamine (Q) at 547, Alanine (A) at551, Alanine (A) at 555, Glutamic acid (E) at 557, Methionine (M) at561, Serine (S) at 563, Glutamine (Q) at 577, Serine (S) at 583, Valine(V) at 593, Threonine (T) at 596, Alanine (A) at 661, Valine (V) at 662,Threonine (T) at 664, Proline (P) at 665, Threonine (T) at 710, AsparticAcid (D) at 717, Asparagine (N) at 718, Glutamic acid (E) at 719, andSerine (S) at 723:

(SEQ ID NO: 59) MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPLGEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASAGSTNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLARTQSTGGTAGTRELLFSQAGPSNMSAQAKNWLPGPCYRQQRVSKTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEDRFFPSSGVLIFGKQGAGANNTALENVMMTSEEEIKTTNPVATEQYGVVASNLQSSNTAPVTGTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPANPPAVFTPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYAKSTNVDFAVDNEGVYSEPRPIGTRYLTRNL.

In other embodiments, a variant AAV capsid protein comprises an aminoacid sequence at least 85%, at least 90%, at least 95% or at least 98%identical to a wild-type AAV capsid sequence selected from the groupconsisting of the ancestral variant disclosed herein as SEQ ID NO:58,comprises one or more amino acid residues selected from the groupconsisting of: Alanine (A) at 264, Alanine (A) at 266, Serine (S) at268, Alanine (A) at 448, Threonine (T) at 459, Arginine (R) at 460,Alanine (A) at 467, Serine (S) at 470, Asparagine (N) at 471, Alanine(A) at 474, Serine (S) at 495, Asparagine (D) at 516, Asparagine (D) at533, Glutamine (Q) at 547, Alanine (A) at 551, Alanine (A) at 555,Glutamic acid (E) at 557, Methionine (M) at 561, Serine (S) at 563,Glutamine (Q) at 577, Serine (S) at 583, Valine (V) at 593, Threonine(T) at 596, Alanine (A) at 661, Valine (V) at 662, Threonine (T) at 664,Proline (P) at 665, Threonine (T) at 710, Aspartic Acid (D) at 717,Asparagine (N) at 718, Glutamic acid (E) at 719, and Serine (S) at 723;and also comprises i) one or more amino acid substitutions selected fromthe group consisting of P34A, S109T+V708I, A593E+N596D, V708I+V719M,V708I+G727D, S109T+A493V+A593E+V708I, V708I+R733C, Q164K, and I698Vand/or (ii) a peptide insertion selected from the group consisting ofQADTTKN (SEQ ID NO:13), ISDQTKH (SEQ ID NO:14), ASDSTKA (SEQ ID NO:15),NQDYTKT (SEQ ID NO:16), HDITKNI (SEQ ID NO:17), PQANANEN (SEQ ID NO:63),TNRTSPD (SEQ ID NO:24), PNSTHGS (SEQ ID NO:25), KDRAPST (SEQ ID NO:26),HQDTTKN (SEQ ID NO:19), HPDTTKN (SEQ ID NO:18), NKTTNKD (SEQ ID NO:20),GKSKVID (SEQ ID NO:23), PISNENEH (SEQ ID NO:64), LAQADTTKNA (SEQ IDNO:27), LAISDQTKHA (SEQ ID NO:28), LGISDQTKHA (SEQ ID NO:29), LAASDSTKAA(SEQ ID NO:30), LAHDITKNIA (SEQ ID NO:32), LPQANANENA (SEQ ID NO:37),LANQDYTKTA (SEQ ID NO:31), LATNRTSPDA (SEQ ID NO:39), LAPNSTHGSA (SEQ IDNO:40), LAKDRAPSTA (SEQ ID NO:41), LAHQDTTKNA (SEQ ID NO:34), LAHPDTTKNA(SEQ ID NO:33), LANKTTNKDA (SEQ ID NO:35), LAGKSKVIDA (SEQ ID NO:38),and LPISNENEHA (SEQ ID NO:36). In some embodiments, the variant AAVcapsid comprises the specified one or more amino acid substitutionsand/or peptide insertions and is otherwise identical to SEQ ID NO:59.

The AAV variants disclosed herein were generated through the use of invivo directed evolution involving the use of primate retinal screensfollowing intravitreal administration. In some embodiments, the variantcapsid proteins disclosed herein, when present in an AAV virion, conferincreased transduction of a retinal cell compared to the transduction ofthe retinal cell by an AAV virion comprising the corresponding parentalAAV capsid protein or wild-type AAV. For example, in some embodiments,the variant capsid proteins disclosed herein, when present in an AAVvirion, confer more efficient transduction of primate retinal cells thanAAV virions comprising the corresponding parental AAV capsid protein orwild-type AAV capsid protein, e.g. the retinal cells take up more AAVvirions comprising the subject variant AAV capsid protein than AAVvirions comprising the parental AAV capsid protein or wild-type AAV. Insome such embodiments, the AAV variant virion or variant rAAV exhibitsat least 2-fold, at least 5-fold, at least 10-fold, at least 15-fold, atleast 20-fold, at least 25-fold, at least 50-fold, or more than 50-fold,increased transduction of a retinal cell, compared to the transductionof the retinal cell by a wild-type AAV virion or rAAV comprising thecorresponding parental AAV capsid protein. In certain such embodiments,the variant capsid proteins disclosed herein, when present in an AAVvirion, confer broader transduction of the primate retinal cells thanAAV virions comprising the corresponding parental AAV capsid protein orwild type AAV capsid protein. In other words, the variant AAV viriontransduces cell types not transduced by virions comprising thecorresponding parental AAV capsid protein, and hence more types of cellsin the retina than the corresponding parental AAV virion. In someembodiments, the AAV variant virion preferentially transduces a retinalcell, e.g., a subject rAAV virion infects a retinal cell with 2-fold,5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 50-fold, or more than50-fold, specificity than another retinal cell or a non-retinal cell,e.g., a cell outside the eye. In some embodiments, the transducedretinal cell is a photoreceptor cell (e.g., rods; cones). In someembodiments, the retinal cell is a retinal ganglion cell (RGC). In someembodiments, the retinal cell is a retinal epithelial cell (RPE cell).In some embodiments, the retinal cell is a Müller glial cell. In someembodiments, the retinal cell is a microglial cell. In some embodiments,the retinal cell is an amacrine cell. In some embodiments, the retinalcell is a bipolar cell. In some embodiments, the retinal cell is ahorizontal cell. An increase in transduction of a retinal cell, e.g.increased efficiency of transduction, broader transduction, morepreferential transduction, etc. may be readily assessed in vitro or invivo by any number of methods in the art for measuring gene expression.For example, the AAV may be packaged with a genome comprising anexpression cassette comprising a reporter gene, e.g. a fluorescentprotein, under the control of a ubiquitous or tissue specific promoter,and the extent of transduction assessed by detecting the fluorescentprotein by, e.g., fluorescence microscopy. As another example, the AAVmay be packaged with a genome comprising a bar coded nucleic acidsequence, and the extent of transduction assessed by detecting thenucleic acid sequence by, e.g., PCR. As another example, the AAV may bepackaged with a genome comprising an expression cassette comprising atherapeutic gene for the treatment of a retinal disease, and the extentof transduction assessed by detecting the treatment of the retinaldisease in an afflicted patient that was administered the AAV.

Ocular diseases that can be treated using a variant rAAV vector orvirion and/or method disclosed herein include, but are not limited to,monogenic diseases, complex genetic diseases, acquired diseases, andtraumatic injuries. Examples of monogenic diseases include, but are notlimited to, Bardet-Biedl syndrome; Batten's Disease; Bietti'sCrystalline Dystrophy; choroideremia; chorioretinal atrophy;chorioretinal degeneration; cone or cone-rod dystrophies (autosomaldominant, autosomal recessive, and X-linked); congenital stationarynight blindness (autosomal dominant, autosomal recessive, and X-linked);disorders of color vision, including achromatopsia (including ACHM2,ACHM3, ACHM4, and ACHM5), protanopia, deuteranopia, and tritanopia;Friedreich's ataxia; Leber's congenital amaurosis (autosomal dominantand autosomal recessive), including, but not limited to, LCA1, LCA2,LCA3, LCA4, LCA6, LCA7, LCA8, LCA12, and LCA15; Leber's Hereditary OpticNeuropathy; macular dystrophy (autosomal dominant and autosomalrecessive), including, but not limited to, acute macular degeneration,Best vitelliform macular dystrophy, pattern dystrophy, North CarolinaMacular Dystrophy, inherited drusen, Sorsby's fundus dystrophy, malattialevantanese, and genetically-determined retinopathy of prematurity;ocular-retinal developmental disease; ocular albinism; optic atrophies(autosomal dominant, autosomal recessive, and X-linked); retinitispigmentosa (autosomal dominant, autosomal recessive, X-linked, andmitochondrially-inherited traits), examples of which include RP1, RP2,RP3, RP10, RP20, RP38, RP40, and RP43; X-linked retinoschisis; Stargardtdisease; and Usher syndrome, including, but not limited to, USH1B,USH1C, USH1D, USH1F, USH1G, USH2A, USH2C, USH2D, AND USH3. Examples ofcomplex genetic diseases include, but are not limited to, glaucoma (openangle, angle-closure, low-tension, normal-tension, congenital,neovascular, pigmentary, pseudoexfoliation); age-related and other formsof macular degeneration, both exudative and non-exudative forms(autosomal dominant and autosomal recessive), such as acute maculardegeneration, vitelliform macular degeneration; retinopathy ofprematurity; and Vogt Koyanagi-Harada (VKH) syndrome. Examples ofacquired diseases include, but are not limited to, acute macularneuroretinopathy; anterior ischemic optic neuropathy and posteriorischemic optic neuropathy; Behcet's disease; branch retinal veinocclusion; choroidal neovascularization; diabetic retinopathy, includingproliferative diabetic retinopathy and associated complications;diabetic uveitis; edema, such as macular edema, cystoid macular edemaand diabetic macular edema; epiretinal membrane disorders; maculartelangiectasia; multifocal choroiditis; non-retinopathy diabetic retinaldysfunction; ocular tumors; optic atrophies; retinal detachment; retinaldisorders, such as central retinal vein occlusion, proliferativevitreoretinopathy (PVR), retinal arterial and venous occlusive disease,vascular occlusion, uveitic retinal disease; uveal effusion; retinalinfective and infiltrative disease; optic nerve diseases such asacquired optic atrophy. Examples of traumatic injuries include, but arenot limited to, histoplasmosis; optic nerve trauma; ocular trauma whichaffects a posterior ocular site or location; retinal trauma; viralinfection of the eye; viral infection of the optic nerve; a posteriorocular condition caused by or influenced by an ocular laser treatment;posterior ocular conditions caused by or influenced by a photodynamictherapy; photocoagulation, radiation retinopathy; and sympatheticophthalmia.

In another embodiment, a variant capsid disclosed herein comprises aheterologous nucleic acid comprising a nucleotide sequence encoding agene product such as, without limitation, an interfering RNA, a longnon-coding RNA, a short non-coding RNA, an antisense RNA, an aptamer, apolypeptide, a secreted antibody, a single chain antibody, a V_(HH)domain, a soluble receptor, an affibody, a knottin, a DARPin, acenturin, a chaperone, a site-specific nuclease that provides forsite-specific knock-down of gene function or a modified site-specificnuclease that provides for gene-specific activation of transcription.

A rAAV variant virion disclosed herein comprises a heterologous nucleicacid comprising a nucleotide sequence encoding a gene product. In someembodiments, the gene product is an interfering RNA. In someembodiments, the gene product is a long non-coding RNA. In someembodiments, the gene product is a short non-coding RNA. In someembodiments, the gene product is an antisense RNA. In some embodiments,the gene product is an aptamer. In some embodiments, the gene product isa polypeptide. In some embodiments, the gene product is a secretedantibody. In some embodiments, the gene product is a single chainantibody. In some embodiments, the gene product is a V_(HH) domain. Insome embodiments, the gene product is a soluble receptor. In someembodiments, the gene product is an affibody. In some embodiments, thegene product is a knottin. In some embodiments, the gene product is aDARPin. In some embodiments, the gene product is a centurin. In someembodiments, the gene product is a chaperone. In some embodiments, thegene product is a site-specific nuclease that provide for site-specificknock-down of gene function.

The uses of the gene product include, but are not limited to, enhancingthe level of a factor in a cell, enhancing the level of a factor in aneighboring cell through secretion of a factor, decreasing the level ofa factor in a cell, or decreasing the level of a factor in a neighboringcell through secretion of a factor. The gene product can be designed tosupplement the level of a defective of missing gene product, decreasethe level of a defective of missing gene product, introduce a newsupporting gene product, supplement the level of a supporting geneproduct, decrease the level of a hindering gene product, or bothdecrease the level of a hindering gene product and introduce orsupplement the level of a supporting gene product.

Gene products delivered by the subject AAV variants can be used to alterthe level of gene products or gene product activity directly orindirectly linked to retinal diseases and trauma. Genes whose geneproducts are directly or indirectly linked to genetic diseases include,e.g., ADP-ribosylation factor-like 6 (ARL6); BBSome interacting protein1 (BBIP1); BBSome protein I (BBS1); BBSome protein 2 (BBS2); BBSomeprotein 4 (BBS4); BBSome protein 5 (BBS5); BBSome protein 7 (BBS7);BBSome protein 9 (BBS9); BBSome protein 10 (BBS10); BBSome protein 12(BBS12); centrosomal protein 290 kDa (CEP290); intraflagellar transportprotein 172 (IFT172); intraflagellar transport protein 27 (IFT27);inositol polyphosphate-5-phosphatase E (INPP5E); inwardly-rectifyingpotassium channel subfamily J member 13 (KCNJ13); leucine zippertranscription factor like-1 (LZTFL1); McKusick-Kaufman syndrome protein(MKKS); Meckel syndrome type 1 protein (MKS1); nephronophthisis 3protein (NPHP1); serologically-defined colon cancer antigen 8 (SDCCAG8);tripartite motif-containing protein 32 (TRIM32); tetratricopeptiderepeat domain 8 (TTC8); Batten disease protein (CLN3); cytochrome P4504V2 (CYP4V2); Rab escort protein 1 (CHM); PR (positive regulatory)domain-containing 13 protein (PRDM13); RPE-retinal G protein-coupledreceptor (RGR); TEA domain family member 1 (TEAD1);arylhydrocarbon-interacting receptor protein-like 1 (AIPL1); cone-rodotx-like photoreceptor homeobox transcription factor (CRX); guanylatecyclase activating protein 1A (GUCA1A); retinal-specific guanylatecyclase (GUCY2D); phosphatidylinositol transfer membrane-associatedfamily member 3 (PITPNM3); prominin 1 (PROM1); peripherin (PRPH);peripherin 2 (PRPH2); regulating synaptic membrane exocytosis protein 1(RIMS1); semaphorin 4A (SEMA4A); human homolog of C. elegans unc119protein (UNC119); ATP-binding cassette transporter—retinal (ABCA4); ADAMmetallopeptidase domain 9 (ADAM9); activating transcription factor 6(ATF6); chromosome 21 open reading frame 2 (C21orf2); chromosome 8 openreading frame 37 (C8orf37); calcium channel; voltage-dependent; alpha2/delta subunit 4 (CACNA2D4); cadherin-related family member 1(protocadherin 21) (CDHR1); ceramide kinase-like protein (CERKL); conephotoreceptor cGMP-gated cation channel alpha subunit (CNGA3); conecyclic nucleotide-gated cation channel beta 3 subunit (CNGB3); cyclin M4(CNNM4); guanine nucleotide binding protein (G protein); alphatransducing activity polypeptide 2 (GNAT2); potassium channel subfamilyV member 2 (KCNV2); Phosphodiesterase 6C (PDE6C); Phosphodiesterase 6H(PDE6H); proteome of centriole 1 centriolar protein B (POC1B); RAB28member of RAS oncogene family (RAB28); retina and anterior neural foldhomeobox 2 transcription factor (RAX2); 11-cis retinol dehydrogenase 5(RDH5); RP GTPase regulator-interacting protein 1 (RPGRIP1); tubulintyrosine ligase-like family member 5 (TTLL5); L-type voltage-gatedcalcium channel alpha-1 subunit (CACNA1F); retinitis pigmentosa GTPaseregulator (RPGR); rod transducin alpha subunit (GNAT1); rod cGMPphosphodiesterase beta subunit (PDE6B); rhodopsin (RHO); calcium bindingprotein 4 (CABP4); G protein-coupled receptor 179 (GPR179); rhodopsinkinase (GRK1); metabotropic glutamate receptor 6 (GRM6); leucine-richrepeat immunoglobulin-like transmembrane domains protein 3 (LRIT3);arrestin (s-antigen) (SAG); solute carrier family 24 (SLC24A1);transient receptor potential cation channel, subfamily M, member 1(TRPM1); nyctalopin (NYX); green cone opsin (OPN1LW); red cone opsin(OPN1MW); blue cone opsin (OPN1SW); frataxin (FXN); inosinemonophosphate dehydrogenase 1 (IMPDH1); orthodenticle homeobox 2 protein(OTX2); crumbs homolog 1 (CRB1); death domain containing protein 1(DTHD1); growth differentiation factor 6 (GDF6); intraflagellartransport 140 Chlamydomonas homolog protein (IFT140); IQ motifcontaining B1 protein (IQCB1); lebercilin (LCA5); lecithin retinolacyltransferase (LRAT); nicotinamide nucleotide adenylyltransferase 1(NMNAT1); RD3 protein (RD3); retinol dehydrogenase 12 (RDH12); retinalpigment epithelium-specific 65 kD protein (RPE65); spermatogenesisassociated protein 7 (SPATA7); tubby-like protein 1 (TULP1);mitochondiral genes (KSS, LHON, MT-ATP6, MT-TH, MT-TL1, MT-TP, MT-TS2,mitochondrially encoded NADH dehydrogenases [MT-ND]); bestrophin 1(BEST1); C1q and tumor necrosis-related protein 5 collagen (CIQTNF5);EGF-containing fibrillin-like extracellular matrix protein 1 (EFEMP1);elongation of very long fatty acids protein (ELOVL4); retinal fascinhomolog 2, actin bundling protein (FSCN2); guanylate cyclase activatingprotein 1B (GUCA1B); hemicentin 1 (HMCN1); interphotoreceptor matrixproteoglycan 1 (IMPG1); retinitis pigmentosa 1-like protein 1 (RP1L1);tissue inhibitor of metalloproteinases-3 (TIMP3); complement factor H(CFH); complement factor D (CFD); complement component 2 (C2);complement component 3 (C3); complement factor B (CFB); DNA-damageregulated autophagy modulator 2 (DRAM2); chondroitin sulfateproteoglycan 2 (VCAN); mitofusin 2 (MFN2); nuclear receptor subfamily 2group F member 1 (NR2F1); optic atrophy 1 (OPA1); transmembrane protein126A (TMEM126A); inner mitochondrial membrane translocase 8 homolog A(TIMM8A); carbonic anhydrase IV (CA4); hexokinase 1 (HK1); kelch-like 7protein (KLHL7); nuclear receptor subfamily 2 group E3 (NR2E3); neuralretina lucine zipper (NRL); olfactory receptor family 2 subfamily Wmember 3 (OR2W3); pre-mRNA processing factor 3 (PRPF3); pre-mRNAprocessing factor 4 (PRPF4); pre-mRNA processing factor 6 (PRPF6);pre-mRNA processing factor 8 (PRPF8); pre-mRNA processing factor 31(PRPF31); retinal outer segment membrane protein 1 (ROM1); retinitispigmentosa protein 1 (RP1); PIM1-kinase associated protein 1 (RP9);small nuclear ribonucleoprotein 200 kDa (SNRNP200); secretedphosphoprotein 2 (SPP2); topoisomerase I binding arginine/serine richprotein (TOPORS); ADP-ribosylation factor-like 2 binding protein(ARL2BP); chromosome 2 open reading frame 71 (C2orf71); clarin-1(CLRN1); rod cGMP-gated channel alpha subunit (CNGA1); rod cGMP-gatedchannel beta subunit (CNGB1); cytochrome P450 4V2 (CYP4V2);dehydrodolichyl diphosphate synthetase (DHDDS); DEAH box polypeptide 38(DHX38); ER membrane protein complex subunit 1 (EMC1); eyesshut/spacemaker homolog (EYS); family with sequence similarity 161member A (FAM161A); G protein-coupled receptor 125 (GPR125);heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT);NAD(+)-specific isocitrate dehydrogenase 3 beta (IDH3B);interphotoreceptor matrix proteoglycan 2 (IMPG2); KIAA1549 protein(KIAA1549); kizuna centrosomal protein (KIZ); male germ-cell associatedkinase (MAK); c-mer protooncogene receptor tyrosine kinase (MERTK);mevalonate kinase (MVK); NIMA (never in mitosis gene A)-related kinase 2(NEK2); neuronal differentiation protein 1 (NEUROD); cGMPphosphodiesterase alpha subunit (PDE6A); phosphodiesterase 6GcGMP-specific rod gamma (PDE6G); progressive rod-cone degenerationprotein (PRCD); retinol binding protein 3 (RBP3); retinaldehyde-bindingprotein 1 (RLBP1); solute carrier family 7 member 14 (SLC7A14); usherin(USH2A); zinc finger protein 408 (ZNF408); zinc finger protein 513(ZNF513); oral-facial-digital syndrome 1 protein (OFD1); retinitispigmentosa 2 (RP2); retinoschisin (RS1); abhydrolase domain containingprotein 12 (ABHD12); cadherin-like gene 23 (CDH23); centrosomal protein250 kDa (CEP250); calcium and integrin binding family member 2 (CIB2);whirlin (DFNB31); monogenic audiogenic seizure susceptibility 1 homolog(GPR98); histidyl-tRNA synthetase (HARS); myosin VIIA (MYO7A);protocadherin 15 (PCDH15); harmonin (USH1C); human homolog of mousescaffold protein containing ankyrin repeats and SAM domain (USH1G);dystrophin (DMD); norrin (NDP); phosphoglycerate kinase (PGK1); calpain5 (CAPN5); frizzled-4 Wnt receptor homolog (FZD4); integral membraneprotein 2B (ITM2B); low density lipoprotein receptor-related protein 5(LRP5); micro RNA 204 (MIR204); retinoblastoma protein 1 (RB1);tetraspanin 12 (TSPAN12); chromosome 12 open reading frame 65(C12orf65); cadherin 3 (CDH3); membrane-type frizzled-related protein(MFRP); ornithine aminotransferase (OAT); phospholipase A2 group V(PLA2G5); retinol-binding protein 4 (RBP4); regulator of G-proteinsignaling 9 (RGS9); regulator of G-protein signaling 9-binding protein(RGS9BP); ARMS2; excision repair cross-complementing rodent repairdeficiency complementation group 6 protein (ERCC6); fibulin 5 (FBLN5);HtrA serine peptidase 1 (HTRA1); toll-like receptor 3 (TLR3); andtoll-like receptor 4 (TLR4).

Genes whose gene products induce or promote apoptosis are referred toherein as “pro-apoptotic genes” and the products of those genes (mRNA;protein) are referred to as “pro-apoptotic gene products.” Pro-apoptotictargets include, e.g., Bax gene products; Bid gene products; Bak geneproducts; Bad gene products; Bcl-2; Bcl-X1. Anti-apoptotic gene productsinclude X-linked inhibitor of apoptosis.

Genes whose gene products induce or promote angiogenesis are referred toherein as “pro-angiogenic genes” and the products of those genes (mRNA;protein) are referred to as “pro-angiogenic gene products.”Pro-angiogenic targets include, e.g., vascular endothelial growth factor(VEGFa, VEGFb, VEGFc, VEGFd); vascular endothelial growth factorreceptor 1 (VEGFR1); vascular endothelial growth factor receptor 2(VEGFR2); Fms-Related Tyrosine Kinase 1 (Flt1); placenta growth factor(PGF); Platelet-derived growth factor (PDGF); angiopoietins; sonichedgehog. Genes whose gene products inhibit angiogenesis are referred toherein as “anti-angiogenic genes” and the products of those genes (mRNA;protein) are referred to as “anti-angiogenic gene products.”Anti-angiogenic gene products include endostatin; tumstatin;angiostatin; pigment epithelium-derived factor (PEDF), and fusionproteins or antibodies that are specific for pro-angiogenic targetsand/or their receptors, e.g. the anti-VEGF fusion proteins sFLT1 orEylea, the VEGF-specific antibodies Lucentis™ and Avastin™, etc.

Genes whose gene products function as immune modulators, e.g.,complement factors, toll-like receptors, are called “immunomodulatorygenes”. Exemplary immunomodulatory genes include cytokines, chemokines,and the fusion proteins or antibodies that are specific for them and/ortheir receptors, e.g. the anti-IL -6 fusion protein Rilonacept™, theComplement Factor H-specific antibody lampamizumab, etc. Genes whosegene products function as neuroprotective factors, e.g., plateletderived growth factor receptor (PDGFR); glial derived neurotrophicfactor (GDNF); rod-derived con viability factor (RdCVF); fibroblastgrowth factor (FGF); neurturin (NTN); ciliary neurotrophic factor(CNTF); nerve growth factor (NGF); neurotrophin-4 (NT4); brain derivedneurotrophic factor (BDNF); epidermal growth factor. Genes whose geneproducts function as light responsive opsins, e.g., opsin; rhodopsin;channel rhodopsin; halo rhodopsin.

In some cases, a gene product of interest is a site-specificendonuclease that provide for site-specific knock-down of gene function,e.g., where the endonuclease knocks out an allele associated with aretinal disease. For example, where a dominant allele encodes adefective copy of a gene that, when wild-type, is a retinal structuralprotein and/or provides for normal retinal function, a site-specificendonuclease can be targeted to the defective allele and knock out thedefective allele.

In addition to knocking out a defective allele, a site-specific nucleasecan also be used to stimulate homologous recombination with a donor DNAthat encodes a functional copy of the protein encoded by the defectiveallele. Thus, e.g., a subject rAAV virion can be used to deliver both asite-specific endonuclease that knocks out a defective allele, and canbe used to deliver a functional copy of the defective allele, resultingin repair of the defective allele, thereby providing for production of afunctional retinal protein (e.g., functional retinoschisin, functionalRPE65, functional peripherin, etc.). See, e.g., Li et al. (2011) Nature475:217. In some embodiments, a rAAV virion disclosed herein comprises aheterologous nucleotide sequence that encodes a site-specificendonuclease; and a heterologous nucleotide sequence that encodes afunctional copy of a defective allele, where the functional copy encodesa functional retinal protein. Functional retinal proteins include, e.g.,retinoschisin, RPE65, retinitis pigmentosa GTPase regulator(RGPR)-interacting protein-1, peripherin, peripherin-2, and the like.

Site-specific endonucleases that are suitable for use include, e.g.,meganucleases; zinc finger nucleases (ZFNs); transcriptionactivator-like effector nucleases (TALENs); and Clustered regularlyinterspaced short palindromic repeats/CRISPR-associated (Cas), wheresuch site-specific endonucleases are non-naturally occurring and aremodified to target a specific gene. Such site-specific nucleases can beengineered to cut specific locations within a genome, and non-homologousend joining can then repair the break while inserting or deletingseveral nucleotides. Such site-specific endonucleases (also referred toas “INDELs”) then throw the protein out of frame and effectively knockout the gene. See, e.g., U.S. Patent Publication No. 2011/0301073.

In some embodiments of the variant rAAV vector disclosed herein, anucleotide sequence encoding a gene product of interest is operablylinked to a constitutive promoter. Suitable constitutive promotersinclude e.g. cytomegalovirus promoter (CMV) (Stinski et al. (1985)Journal of Virology 55(2): 431-441), CMV early enhancer/chicken β-actin(CBA) promoter/rabbit β-globin intron (CAG) (Miyazaki et al. (1989) Gene79(2): 269-277, CB^(SB) (Jacobson et al. (2006) Molecular Therapy 13(6):1074-1084), human elongation factor 1α promoter (EF1α) (Kim et al.(1990) Gene 91(2): 217-223), human phosphoglycerate kinase promoter(PGK) (Singer-Sam et al. (1984) Gene 32(3): 409-417, mitochondrialheavy-strand promoter (Loderio et al. (2012) PNAS 109(17): 6513-6518),ubiquitin promoter (Wulff et al. (1990) FEBS Letters 261: 101-105). Inother embodiments, a nucleotide sequence encoding a gene product ofinterest is operably linked to an inducible promoter. In some instances,a nucleotide sequence encoding a gene product of interest is operablylinked to a tissue-specific or cell type-specific regulatory element.For example, in some instances, a nucleotide sequence encoding a geneproduct of interest is operably linked to a photoreceptor-specificregulatory element (e.g., a photoreceptor-specific promoter), e.g., aregulatory element that confers selective expression of the operablylinked gene in a photoreceptor cell. Suitable photoreceptor-specificregulatory elements include, e.g., a rhodopsin promoter; a rhodopsinkinase promoter (Young et al. (2003) Ophthalmol. Vis. Sci. 44:4076); abeta phosphodiesterase gene promoter (Nicoud et al. (2007) J. Gene Med.9:1015); a retinitis pigmentosa gene promoter (Nicoud et al. (2007)supra); an interphotoreceptor retinoid-binding protein (IRBP) geneenhancer (Nicoud et al. (2007) supra); an IRBP gene promoter (Yokoyamaet al. (1992) Exp Eye Res. 55:225), an opsin gene promoter (Tucker etal. (1994) PNAS 91:2611-2615), a retinoschisin gene promoter (Park etal. (2009) Gene Therapy 16(7): 916-926), a CRX homeodomain protein genepromoter (Furukawa et al. (2002) The Journal of Neuroscience 22(5):1640-1647), a guanine nucleotide binding protein alpha transducingactivity polypeptide 1 (GNAT1) gene promoter (Lee et al. (2010) GeneTherapy 17:1390-1399), a neural retina-specific leucine zipper protein(NRL) gene promoter (Akimoto et al. (2006) PNAS 103(10): 3890-3895),human cone arrestin (hCAR) promoter (Li et al. (2002) Biochemistry andMolecular Biology 43: 1375-1383), and the PR2.1, PR1.7, PR1.5, and PR1.1promoters (Ye et al. (2016) Human Gene Therapy 27(1): 72-82)). In someinstances, a nucleotide sequence encoding a gene product of interest isoperably linked to a retinal pigment epithelia (RPE) cell-specificregulatory element (e.g., a RPE-specific promoter), e.g., a regulatoryelement that confers selective expression of the operably linked gene ina RPE cell. Suitable RPE-specific regulatory elements include, e.g., anRPE65 gene promoter (Meur et al. (2007) Gene Therapy 14: 292-303), acellular retinaldehyde-binding protein (CRALBP) gene promoter (Kennedyet al. (1998) Journal of Biological Chemistry 273: 5591-5598), a pigmentepithelium-derived factor (PEDF aka serpin F1) gene promoter (Kojima etal. (2006) Molecular and Cellular Biochemistry 293(1-2): 63-69), and avitelliform macular dystrophy (VMD2) promoter (Esumi et al. (2004) TheJournal of Biological Chemistry 279(18): 19064-19073). In someinstances, a nucleotide sequence encoding a gene product of interest isoperably linked to a Müller glia cell-specific regulatory element (e.g.,a glial-specific promoter), e.g., a regulatory element that confersselective expression of the operably linked gene in a retinal glialcell. Suitable glial-specific regulatory elements include, e.g., a glialfibrillary acidic protein (GFAP) promoter (Besnard et al. (1991) Journalof Biological Chemistry 266(28): 18877-18883). In some instances, anucleotide sequence encoding a gene product of interest is operablylinked to a bipolar cell-specific regulatory element (e.g., abipolar-specific promoter), e.g., a regulatory element that confersselective expression of the operably linked gene in a bipolar cell.Suitable bipolar-specific regulatory elements include, e.g., a GRM6promoter (Cronin et al. (2014) EMBO Molecular Medicine 6(9): 1175-1190).

For the purposes of the invention, the disclosure herein provides anisolated nucleic acid comprising a nucleotide sequence that encodes avariant AAV capsid protein as described above. An isolated nucleic acidcan be an AAV vector, e.g., a recombinant AAV vector.

The disclosure herein also provides a method of treating a retinaldisease, the method comprising administering to an individual in needthereof an effective amount of a rAAV variant virion comprising atransgene of interest as described above and disclosed herein. One ofordinary skill in the art would be readily able to determine aneffective amount of the subject rAAV virion and that the disease hadbeen treated by testing for a change in one or more functional oranatomical parameters, e.g. visual acuity, visual field,electrophysiological responsiveness to light and dark, color vision,contrast sensitivity, anatomy, retinal health and vasculature, ocularmotility, fixation preference, and stability.

Nonlimiting methods for assessing retinal function and changes thereofinclude assessing visual acuity (e.g. best-corrected visual acuity[BCVA], ambulation, navigation, object detection and discrimination),assessing visual field (e.g. static and kinetic visual field perimetry),performing a clinical examination (e.g. slit lamp examination of theanterior and posterior segments of the eye), assessingelectrophysiological responsiveness to all wavelengths of light and dark(e.g. all forms of electroretinography (ERG) [full-field, multifocal andpattern], all forms of visual evoked potential (VEP), electrooculography(EOG), color vision, dark adaptation and/or contrast sensitivity).Nonlimiting methods for assessing anatomy and retinal health and changesthereof include Optical Conherence Tomography (OCT), fundus photography,adaptive optics scanning laser ophthalmoscopy (AO-SLO), fluorescenceand/or autofluorescence; measuring ocular motility and eye movements(e.g. nystagmus, fixation preference, and stability), measuring reportedoutcomes (patient-reported changes in visual and non-visually-guidedbehaviors and activities, patient-reported outcomes [PRO],questionnaire-based assessments of quality-of-life, daily activities andmeasures of neurological function (e.g. functional Magnetic ResonanceImaging (MRI)).

In some embodiments, an effective amount of the subject rAAV virionresults in a decrease in the rate of loss of retinal function,anatomical integrity, or retinal health, e.g. a 2-fold, 3-fold, 4-fold,or 5-fold or more decrease in the rate of loss and hence progression ofdisease, for example, a 10-fold decrease or more in the rate of loss andhence progression of disease. In some embodiments, the effective amountof the subject rAAV virion results in a gain in visual function, retinalfunction, an improvement in retinal anatomy or health, and/or animprovement in ocular motility and/or improvement in neurologicalfunction, e.g. a 2-fold, 3-fold, 4-fold or 5-fold improvement or more inretinal function, retinal anatomy or health, and/or improvement inocular motility, e.g. a 10-fold improvement or more in retinal function,retinal anatomy or health, and/or improvement in ocular motility. Aswill be readily appreciated by the ordinarily skilled artisan, the doserequired to achieve the desired treatment effect will typically be inthe range of 1×10⁸ to about 1×10¹⁵ recombinant virions, typicallyreferred to by the ordinarily skilled artisan as 1×10⁸ to about 1×10¹⁵“vector genomes”.

A subject rAAV virion can be administered via intraocular injection, forexample by intravitreal injection, by subretinal injection, bysuprachoroidal injection, or by any other convenient mode or route ofadministration that will result in the delivery of the rAAV virion tothe eye. Other convenient mancestodes or routes of administrationinclude, without limitation, intravenous, intra-arterial, peri-ocular,intracameral, subconjunctival and sub-tenons injections and topicaladministration and intranasal. When administered via intravitrealinjection, the subject rAAV virion is able to move through the vitreousand traverse the internal limiting membrane (also referred to herein asan inner limiting membrane, or “ILM”; a thin, transparent acellularmembrane on the surface of the retina forming the boundary between theretina and the vitreous body, formed by astrocytes and the end feet ofMüller cells), and/or moves through the layers of the retina moreefficiently, compared to the capability of an AAV virion comprising thecorresponding parental AAV capsid protein.

A variant capsid protein disclosed herein is isolated, e.g., purified.In some embodiments, a variant capsid protein disclosed herein isincluded in an AAV vector or a recombinant AAV (rAAV) virion. In otherembodiments, such AAV variant vectors and/or AAV variant virions areused in an in vivo or ex vivo method of treating ocular disease in theprimate retina.

The disclosure herein further provides host cells such as, withoutlimitation, isolated (genetically modified) host cells comprising asubject nucleic acid. A host cell according to the invention disclosedherein, can be an isolated cell, such as a cell from an in vitro cellculture. Such a host cell is useful for producing a subject rAAV variantvirion, as described herein. In one embodiment, such a host cell isstably genetically modified with a nucleic acid. In other embodiments, ahost cell is transiently genetically modified with a nucleic acid. Sucha nucleic acid is introduced stably or transiently into a host cell,using established techniques, including, but not limited to,electroporation, calcium phosphate precipitation, liposome-mediatedtransfection, and the like. For stable transformation, a nucleic acidwill generally further include a selectable marker, e.g., any of severalwell-known selectable markers such as neomycin resistance, and the like.Such a host cell is generated by introducing a nucleic acid into any ofa variety of cells, e.g., mammalian cells, including, e.g., murinecells, and primate cells (e.g., human cells). Exemplary mammalian cellsinclude, but are not limited to, primary cells and cell lines, whereexemplary cell lines include, but are not limited to, 293 cells, COScells, HeLa cells, Vero cells, 3T3 mouse fibroblasts, C3H10T1/2fibroblasts, CHO cells, and the like. Exemplary host cells include,without limitation, HeLa cells (e.g., American Type Culture Collection(ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096),293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g.,ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651),RAT1 cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney(HEK) cells (ATCC No. CRL1573), HLHepG2 cells, and the like. A host cellcan also be made using a baculovirus to infect insect cells such as Sf9cells, which produce AAV (see, e.g., U.S. Pat. No. 7,271,002; U.S.patent application Ser. No. 12/297,958). In some embodiments, agenetically modified host cell includes, in addition to a nucleic acidcomprising a nucleotide sequence encoding a variant AAV capsid protein,as described above, a nucleic acid that comprises a nucleotide sequenceencoding one or more AAV rep proteins. In other embodiments, a host cellfurther comprises an rAAV variant vector. An rAAV variant virion can begenerated using such host cells. Methods of generating an rAAV virionare described in, e.g., U.S. Patent Publication No. 2005/0053922 andU.S. Patent Publication No. 2009/0202490.

The disclosure herein additionally provides a pharmaceutical compositioncomprising: a) the rAAV variant virion, as described above and disclosedherein; and b) a pharmaceutically acceptable carrier, diluent,excipient, or buffer. In some embodiments, the pharmaceuticallyacceptable carrier, diluent, excipient, or buffer is suitable for use ina human or non-human patient. Such excipients, carriers, diluents, andbuffers include any pharmaceutical agent that can be administeredwithout undue toxicity. Pharmaceutically acceptable excipients include,but are not limited to, liquids such as water, saline, glycerol andethanol. Pharmaceutically acceptable salts can be included therein, forexample, mineral acid salts such as hydrochlorides, hydrobromides,phosphates, sulfates, and the like; and the salts of organic acids suchas acetates, propionates, malonates, benzoates, and the like.Additionally, auxiliary substances, such as wetting or emulsifyingagents, surfactants, pH buffering substances, and the like, may bepresent in such vehicles. A wide variety of pharmaceutically acceptableexcipients are known in the art and need not be discussed in detailherein. Pharmaceutically acceptable excipients have been amply describedin a variety of publications, including, for example, A. Gennaro (2000)“Remington: The Science and Practice of Pharmacy,” 20th edition,Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and DrugDelivery Systems (1999) H. C. Ansel et al., eds., 7^(th) ed.,Lippincott, Williams, & Wilkins; and Handbook of PharmaceuticalExcipients (2000) A. H. Kibbe et al., eds., 3^(rd) ed. Amer.Pharmaceutical Assoc. In some aspects of the present invention, thepresent invention provides a pharmaceutical composition comprising about1×10⁸ to about 1×10¹⁵ recombinant viruses or 1×10⁸ to about 1×10¹⁵vector genomes, wherein each said recombinant virus comprises a genomeencoding one or more gene products.

The following examples are set forth to provide the ordinarily skilledartisan with a complete disclosure and description for guidance as tohow to make and use the variant AAV capsids disclosed herein, and arenot intended to limit the scope of the invention disclosed herein. Inaddition, the following examples are not intended to represent that theexperiments below are all or the only experiments performed.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

General methods in molecular and cellular biochemistry can be found insuch standard textbooks as Molecular Cloning: A Laboratory Manual, 3rdEd. (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols inMolecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); NonviralVectors for Gene Therapy (Wagner et al. eds., Academic Press 1999);Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); ImmunologyMethods Manual (I. Lefkovits ed., Academic Press 1997); and Cell andTissue Culture: Laboratory Procedures in Biotechnology (Doyle &Griffiths, John Wiley & Sons 1998), the disclosures of which areincorporated herein by reference. Reagents, cloning vectors, and kitsfor genetic manipulation referred to in this disclosure are availablefrom commercial vendors such as BioRad, Stratagene, Invitrogen,Sigma-Aldrich, and ClonTech.

Example 1

Intravitreal Injection and Tissue Harvesting. A single male cynomolgusmacaque (Macaca fascicularis) age 4-10 years old and weighing at least 4kg was dosed via intravitreal injection through the sclera(approximately 3 mm behind the limbus using a procedure and deliverydevice suitable for human use). The animal was anesthetized and giventhe topical anesthetic 100 μL of the library was administered to eacheye

Euthanasia was performed by trained veterinary staff using 100 mg/kgpentobarbital sodium intravenous injection on day 14±3. Eyes werenucleated and stored at 4° C. until dissection.

Tissue Dissection. Eyes were cut along the Ora serrata with a scalpel,and the anterior segment was removed. Relief cuts were made into theretina around the fovea to enable flat mounting of the retina, and thevitreous was removed. Six samples of the retina from each quadrant(superior, inferior, nasal, and temporal) were collected, as shown inFIG. 2, and cellular material corresponding to RPE cells,photoreceptors, biopolar cells, amacrine cells, horizontal cells, and/organglion cells was isolated.

Directed Evolution. The directed evolution process is shown in FIG.1A-1E. Briefly, a viral capsid library comprising 20+ proprietarycombinations of DNA mutation technique and cap genes is created (FIG.1A). Viruses are then packaged (FIG. 1B)—such that each particle iscomposed of a mutant capsid surrounding the cap gene encoding thatcapsid—and purified. The capsid library is placed under selectivepressure in vivo. The tissue or cellular material of interest isharvested to isolate AAV variants that have successfully infected thattarget, and the successful viruses are recovered. Successful clones areenriched through repeated selection (Stage 1—FIG. 1D). Selected capgenes then undergo proprietary re-diversification and are enrichedthrough further selection steps to iteratively increase viral fitness(Stage 2—FIG. 1D). Variants identified during Vector Selection Stages 1and 2 demonstrate the ability to transduce primate retina cells (FIG.1E).

Successful Recovery of AAV Capsid Genomes: Rounds 1-6. The capsidsrecovered from each round of selection were used to package the libraryinjected to initiate the subsequent round of selection. Recovery ofcapsid genes from tissue represents successful internalization oflibrary vectors into the tissue of interest. Following Round 4,additional re-diversification of the library was incorporated prior tolibrary packaging and injection for Round 5. Recovery of viral genomesfrom RPE, PR, inner nuclear layer (INL), and ganglion cell layer (GCL)retinal tissue from a representative round of selection are shown inFIG. 3. Bands within boxes represent successful recovery of viralgenomes.

Sequencing Analysis: Rounds 3-6. During rounds 3-6, sequencing wasperformed on individual clones within the library to determine thefrequency of variants within the population. Variants were evaluated forthe presence of motifs within the sequencing data. Variants were groupedinto motifs based on the presence of a unifying variation (for example,a specific point mutation or specific peptide insertion sequence in aconsistent location within the capsid) that occurred in multiplesequences. Motifs representing at least 5% of the sequenced populationin two or more rounds of the selection or at least 10% of the sequencedpopulation in one or more rounds of the selection are represented inFIG. 4A (Round 3 sequencing analysis), 4B (Round 4 sequencing analysis),4C (Round 5 sequencing analysis), and 4D (Round 6 sequencing analysis).

Several representative clones that were identified as conferringincreased infectivity of retinal cells are listed in Table 1 below (eachclone contains the identified substitution(s) and/or peptide insertionand is otherwise identical to SEQ ID NO:2; the selection round, numberof sequences and frequency (in parentheses) are listed for each clone):

TABLE 1 Amino acid sequence modifications to the AAV VPI capsid proteinthat confer increased infectivity of one or more cells of the retina.Substitutions listed in column 2 are based on the amino acid sequencefor wild type AAV2, i.e. in the absence of inserted peptide. Pan-Insertion Substitution Retinal RPE Photoreceptor 588~LAISDQTKHA~ NoneRound 4, 5, Round 3, Round 4, (SEQ ID NO: 28) 6 5, 6 5 588~LAISDQTKHA~+M1L+L15P Round 6 (SEQ ID NO: 28) +P535S 1 (1.61%) 588~LAISDQTKHA~ +P34ARound 5 (SEQ ID NO: 28) 1 (1.89%) 588~LAISDQTKHA~ +P34A+S731L Round 5(SEQ ID NO: 28) 1 (1.82%) 588~LAISDQTKHA~ +N57D Round 4 (SEQ ID NO: 28)1 (1.33%) 588~LAISDQTKHA~ +N66K Round 5 (SEQ ID NO: 28) 1 (1.82%)588~LAISDQTKHA~ +R81Q Round 6 (SEQ ID NO: 28) 1 (1.61%) 588~LAISDQTKHA~+Q101R Round 4 (SEQ ID NO: 28) 1 (2.27%) 588~LAISDQTKHA~ +S109T Round 3(SEQ ID NO: 28) 1 (1.85%) 588~LAISDQTKHA~ +R144K Round 5 (SEQ ID NO: 28)1 (1.82%) 588~LAISDQTKHA~ +R144M Round 5 (SEQ ID NO: 28) 1 (1.82%)588~LAISDQTKHA~ +Q164K Round 4 (SEQ ID NO: 28) 1 (2.27%) 588~LAISDQTKHA~+Q164K+V708I Rounds 3 (SEQ ID NO: 28) and 4 2 (3.7%) 1 (1.33%)588~LAISDQTKHA~ +T176P Round 4 (SEQ ID NO: 28) 1 (1.33%) 588~LAISDQTKHA~+L188I Round 5 (SEQ ID NO: 28) 1 (2.27%) 588~LAISDQTKHA~ +S196Y Round 4(SEQ ID NO: 28) 1 (1.33%) 588~LAISDQTKHA~ +G226E Round 4 (SEQ ID NO: 28)1 (2.27%) 588~LAISDQTKHA~ +G236V Round 4 (SEQ ID NO: 28) 1 (1.33%)588~LAISDQTKHA~ +I240T Round 3 (SEQ ID NO: 28) 1 (1.85%) 588~LAISDQTKHA~+N312K Round 4 (SEQ ID NO: 28) 1 (1.33%) 588~LAISDQTKHA~ +P363L Round 6(SEQ ID NO: 28) 1 (1.61%) 588~LAISDQTKHA~ +T456K Round 6 (SEQ ID NO: 28)1 (1.61%) 588~LAISDQTKHA~ +I698V Round 5 (SEQ ID NO: 28) 1 (1.89%)588~LAISDQTKHA~ +V708I Round 4, 5, Round 3,  Round 4,  (SEQ ID NO: 28) 64, 5 5 588~LAISDQTKHA~ +V708I+R484C Round 5 (SEQ ID NO: 28) 1 (2.27%)588~LAISDQTKHA~ N312K+N449D+ Engineered Engineered (SEQ ID NO: 28)N551S+I698V+L735Q 588~LGISDQTKHA~ None Round 5 (SEQ ID NO: 29) 1 (1.89%)588~LAQADTTKNA~ None Round 3, 4, Round 3, Round 4, (SEQ ID NO: 27) 5, 64, 5 5 588~LAQADTTKNA~ +E36D Round 5 (SEQ ID NO: 27) 1 (2.27%)588~LAQADTTKNA~ +P250S Round 6 (SEQ ID NO: 27) 1 (1.61%) 588~LAQADTTKNA~+A524T Round 3 (SEQ ID NO: 27) 1 (1.85%) 588~LAQADTTKNA~ +A593E Round 4(SEQ ID NO: 27) 1 (1.33%) 588~LAQADTTKNA~ +I698V Rounds 5(SEQ ID NO: 27) and 6 1 (1.61%) 1 (1.89%) 588~LAQADTTKNA~ +V708IRound 3, 4, Round 3, (SEQ ID NO: 27) 5, 6 4, 5 588~LAQADTTKNA~+V708I+V719M Rounds 3 (SEQ ID NO: 27) and 4 1 (2.08%) 2 (4.55%)588~LAQADTTKNA~ +V719M Round 4 (SEQ ID NO: 27) 1 (2.27%) 588~LAHQDTTKNA~None Round 5 (SEQ ID NO: 34) 2 (3.77%) 588~LANQDYTKTA~ None Rounds 3, 4Rounds 3, Round 5 (SEQ ID NO: 31) and 5 4 and 5 1 (1.82%) 5 (9.26%)2 (4.17%) 1 (1.33%) 2 (4.55%) 3 (5.66%) 2 (2.27%) 588~LANQDYTKTA~+S109T+S463Y Round 3 (SEQ ID NO: 31) 1 (1.85%) 588~LANQDYTKTA~ +D368HRound 3 (SEQ ID NO: 31) 1 (1.85%) 588~LANQDYTKTA~ +V708I Round 4 Round 3(SEQ ID NO: 31) 1 (1.33%) 1 (2.08%) 588~LAHDITKNIA~ None Rounds 3, 4(SEQ ID NO: 32) 5 and 6 1 (1.85%) 1 (1.33%) 1 (1.89%) 2 (3.23%)588~LAHDITKNIA~ +S109T Round 4 (SEQ ID NO: 32) 1 (1.33%) 588~LAHDITKNIA~+R389S Round 5 (SEQ ID NO: 32) 1 (1.89%) 588~LAHDITKNIA~ +A593E Round 3(SEQ ID NO: 32) 1 (1.85%) 588~LAHDITKNIA~ +V708I Round 4, 5(SEQ ID NO: 32) 588~IAHDITKNIA~ +V708I Round 3 (SEQ ID NO: 60) 1 (2.08%)588~LAPNSTHGSA~ +V708I Round 3 (SEQ ID NO: 40) 1 (1.85%) 588~LANKTTNKDA~None Round 5 (SEQ ID NO: 35) 1 (2.27%) 588~LANKTTNKDA~ +N449D Round 4(SEQ ID NO: 35) 1 (2.17%) 588~LAHPDTTKNA~ Round 6 (SEQ ID NO: 33)1 (1.61%) 588~LATNRTSPDA~ Round 6 (SEQ ID NO: 39) 1 (1.61%)588~LPQANANENA~ Round 5 (SEQ ID NO: 37) 1 (1.89%) 588~LAASDSTKAA~Rounds 3 (SEQ ID NO: 30) and 4 1 (1.85%) 1 (1.33%) 588~LAKDRAPSTA~Round 3 (SEQ ID NO: 41) 1 (1.85%) 588~LPISNENEHA~ Round 4(SEQ ID NO: 36) 1 (2.17%) 588~LAGKSKVIDA~ Round 5 (SEQ ID NO: 38)1 (1.82%) NONE P34A Round 5 Rounds 4, Round 4 1 (1.89%) 5 1 (2.17%)3 (6.82%) 1 (2.27%) NONE P64S Round 3 Round 4 1 (1.85%) 1 (2.27%) NONES109T Round 4 Rounds 3, 2 (2.67%) 4, 5 4 (8.33%) 1 (2.27%) 1 (2.27%)NONE S109T+P8L Round 3 1 (2.08%) NONE S109T+Q120R Round 3 1 (2.08%) NONES109T+A493V Round 3 +A593E+V708I 1 (1.85%) NONE Q164K Rounds 4 Round 4and 5 1 (2.27%) 2 (2.67%) 1 (1.89%) NONE Q175H Round 3 Round 4 1 (1.85%)1 (2.17%) NONE S196Y Round 3 Round 4 1 (1.85%) 1 (2.27%) NONE A593ERounds 3, 4, Rounds 3, Round 4 5 4, 5 1 (2.17%) 3 (5.56%) 12 (25%)7 (9.33%) 7 (15.9%) 1 (1.89%) 14 (31.8%) NONE A593E+Q464R Round 31 (2.08%) NONE A593E+N596D Round 4 1 (2.27%) NONE A593E+N596D Round 3+T491A 2 (4.17%) NONE A593E+V708I Rounds 3, 4 Rounds 3, 2 (3.7%) 4, 52 (2.67%) 4 (8.33%) 1 (2.27%) 1 (2.27%) NONE I698V Round 5 Round 51 (1.89%) 1 (2.27%) NONE V708I Rounds 3, 4, Rounds 3, 2 (3.7%) 4, 55 (6.67%) 1 (2.08%) 4 (9.09%) 4 (9.09%) NONE V708I+V719M Round 42 (4.55%) NONE V708I+G727D Round 5 1 (2.27%) NONE V708I+R733C Round 41 (2.17%)

Also identified as a capsid having increased infectivity of one or morecells of the retina was a clone having the following ancestral VP1capsid sequence:

(SEQ ID NO: 59) MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPLGEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASAGSTNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLARTQSTGGTAGTRELLFSQAGPSNMSAQAKNWLPGPCYRQQRVSKTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEDRFFFSSGVLIFGKQGAGANNTALENVMMTSEEEIKTTNPVATEQYGVVASNLQSSNTAPVTGTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPANPPAVFTPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYAKSTNVDFAVDNEGVYSEPRPIGTRYLTRNL. 

This ancestral capsid variant is evolved from the ancestral capsid SEQID NO:58, in which the positions of degeneracy (residues 264, 266, 268,448, 459, 460, 467, 470, 471, 474, 495, 516, 533, 547, 551, 555, 557,561, 563, 577, 583, 593, 596, 661, 662, 664, 665, 710, 717, 718, 719,723) evolved to comprise Alanine (A) at 264, Alanine (A) at 266, Serine(S) at 268, Alanine (A) at 448, Threonine (T) at 459, Arginine (R) at460, Alanine (A) at 467, Serine (S) at 470, Asparagine (N) at 471,Alanine (A) at 474, Serine (S) at 495, Asparagine (D) at 516, Asparagine(D) at 533, Glutamine (Q) at 547, Alanine (A) at 551, Alaninet (A) at555, Glutamic acid (E) at 557, Methionine (M) at 561, Serine (S) at 563,Glutamine (Q) at 577, Serine (S) at 583, Valine (V) at 593, Threonine(T) at 596, Alanine (A) at 661, Valine (V) at 662, Threonine (T) at 664,Proline (P) at 665, Threonine (T) at 710, Aspartic Acid (D) at 717,Asparagine (N) at 718, Glutamic acid (E) at 719, and Serine (S) at 723.

The AAV variant virions disclosed herein may incorporate reasonablerational design parameters, features, modifications, advantages, andvariations that are readily apparent to those skilled in the art in thefield of engineering AAV viral vectors.

Example 2

Directed evolution was employed to discover novel adeno-associated virus(AAV) variants with superior gene delivery to retinal cells followingintravitreal (IVT) administration, a route of administration withsignificant advantages over other methods of gene delivery to the humaneye (Example 1). The cell tropism following intravitreal administrationof the novel AAV variant comprising a P34A substitution and the peptideLAISDQTKHA (SEQ ID NO:28) inserted at amino acid 588 (LAISDQTKHA+P34A)was assessed in vivo in non-human primates (NHP) as a representativeexample of the ability of ISDQTKH (SEQ ID NO:14)-containing AAV variantsto transduce retinal cells.

Recombinant AAV virions comprising either an AAV2 capsid or the novelvariant capsid LAISDQTKHA+P34A and a genome comprising a greenfluorescent protein (GFP) transgene operably linked to a CMV promoter(AAV2.CMV.GFP and LAISDQTKHA+P34A.CMV.GFP, respectively) or a CAGpromoter (AAV2.CAG.EGFP and LAISDQTKHA+P34A.CAG.EGFP, respectively) weremanufactured using standard methods. African Green Monkeys (FIGS. 7, 8)or Cynomolgus macaques (FIG. 9) were injected intravitreally withvarious doses of vector ranging from 4×10¹⁰ vg to 1×10¹² 1e12 vg per eye(see figure legends for details) and the transduction of retinal cellswas assessed in life by fundus fluorescence imaging with a HeidelbergSpectralis™.

Intravitreal delivery of AAVs comprising the novel variantLAISDQTKHA+P34A resulted in broader and more robust transgenicexpression across the NHP retina than AAV2 (FIGS. 7-9). Images revealthat the novel AAV variant capsid provides for robust expression withinthe center of the fovea (an area rich in cones); in the parafoveal ring(an area rich in retinal ganglion cells), and in the periphery (an arearich in many types of cells including rods, Muller glia, amacrine cells,bipolar cells) as early as 2 weeks after injection. In contrast, andconsistent with results reported by others, wild type AAV2 provides forweaker expression that is primarily in the parafoveal ring and can onlybe detected at later time points. Immunohistochemical analysis ofvarious regions of the retina performed 3 weeks after injectionconfirmed that many types of retinal cells, including retinal pigmentepithelial cells, rod and cone photoreceptors, and retinal ganglioncells, had been successfully transduced throughout the retina (FIGS.10A-10E).

This study in illustrates superior gene delivery by theISDQTKH-comprising variant following a clinically preferred route ofadministration as compared to the clinically relevant AAV2. Similarefficacy is achievable with other variants comprising this peptideinsertion motif. Likewise, similar efficacy is achievable with othervariants disclosed herein that were identified using the same directedevolution approach.

Example 3

The cell tropism of the novel AAV variant LAISDQTKHA+P34A for retinalpigment epithelial (RPE) cells and photoreceptor (PR) cells was assessedin vitro in use RPE cells and PR cells generated from fibroblast-derivedhuman induced pluripotent stem cells (FB-iPSC) or human embryonic stemcells (ESC).

AAV virions comprising either an AAV2 capsid or the novel variant capsidLAISDQTKHA+P34A and a genome comprising a green fluorescent protein(EGFP) transgene operably linked to a CAG promoter (AAV2.CAG.EGFP andLAISDQTKHA+P34A.CAG.EGFP, respectively) were manufactured using standardmethods. Human RPE cell cultures were generated from the human embryonicstem cell line ESI-017 or human fibroblast-derived induced pluripotentstem cells (“FB-iPSC”) using a 45-day differentiation protocol.Maturation into RPE cells was confirmed by detecting the expression ofmature RPE markers including RPE65 and BEST1; the synthesis of VEGF andPEDF; and the ability to phagocytose rod outer segments. PR cultureswere generated by a multi-step eye cup formation paradigm and confirmedto comprise PRs by detecting the expression of Recoverin and S Opsinafter 179 days in culture.

Relative to AAV2, LAISDQTKHA+P34A provided for significantly highertransduction efficiency of and transgene expression in human RPEcultures seven days post-infection as determined by immunofluorescence(FIGS. 11A-B), flow cytometry (2.7-fold increase; FIGS. 11C-D) andWestern blot analysis (FIGS. 11E-F). Robust transduction and expressionwas likewise achieved using LAISDQTKHA+P34A.CAG.EGFP in human PRcultures by 32 days post-infection. This study illustrates the superiorability of ISDQTKH (SEQ ID NO:14)-comprising variants to deliver genesto retinal cells.

The preceding merely illustrates the principles of the invention. Itwill be appreciated that those skilled in the art will be able to devisevarious arrangements which, although not explicitly described or shownherein, embody the principles of the invention and are included withinits spirit and scope. Furthermore, all examples and conditional languagerecited herein are principally intended to aid the reader inunderstanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions.

Moreover, all statements herein reciting principles, aspects, andembodiments of the invention as well as specific examples thereof, areintended to encompass both structural and functional equivalentsthereof. Additionally, it is intended that such equivalents include bothcurrently known equivalents and equivalents developed in the future,i.e., any elements developed that perform the same function, regardlessof structure. The scope of the present invention, therefore, is notintended to be limited to the exemplary embodiments shown and describedherein. Rather, the scope and spirit of the present invention isembodied by the appended claims.

We claim:
 1. A method for treating a human subject with X-linked retinitis pigmentosa, the method comprising administering to the subject by intravitreal injection, a pharmaceutical composition comprising a therapeutically effective amount of an infectious recombinant adeno-associated virus (rAAV) virion comprising (i) a variant AAV capsid protein comprising the amino acid sequence set forth in SEQ ID NO:42 and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding a retinitis pigmentosa GTPase regulator protein, said nucleotide sequence operably linked to an ubiquitous promoter or a rhodopsin kinase promoter that directs expression of the retinitis pigmentosa GTPase regulator protein in one or more photoreceptor cells, thereby treating X-linked retinitis pigmentosa in said subject.
 2. The method according to claim 1, wherein said nucleotide sequence is operably linked to a rhodopsin kinase promoter.
 3. The method according to claim 1, comprising administering to the subject a pharmaceutical composition comprising from 10¹¹ to 10¹⁵ vg of said rAAV and a pharmaceutically acceptable excipient.
 4. A method for treating a human subject with X-linked retinitis pigmentosa, the method comprising administering to the subject by subretinal injection, a pharmaceutical composition comprising a therapeutically effective amount of an infectious recombinant adeno-associated virus (rAAV) virion comprising (i) a variant AAV capsid protein comprising the amino acid sequence set forth in SEQ ID NO:42 and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding a retinitis pigmentosa GTPase regulator protein, said nucleotide sequence operably linked to an ubiquitous promoter or a rhodopsin kinase promoter that directs expression of the retinitis pigmentosa GTPase regulator protein in one or more photoreceptor cells, thereby treating X-linked retinitis pigmentosa in said subject.
 5. The method according to claim 4, wherein said nucleotide sequence is operably linked to a rhodopsin kinase promoter.
 6. The method according to claim 4, comprising administering to the subject a pharmaceutical composition comprising from 10¹¹ to 10¹⁵ vg of said rAAV and a pharmaceutically acceptable excipient. 